Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway.

Parkinson's disease is characterized by the deposition of α-synuclein, which leads to synaptic dysfunction, the loss of neuronal connections and ultimately progressive neurodegeneration. Despite extensive research into Parkinson's disease pathogenesis, the mechanisms underlying α-synuclein-mediated synaptopathy have remained elusive. Several lines of evidence suggest that altered nicotinamide adenine dinucleotide (NAD+) metabolism might be causally related to synucleinopathies, including Parkinson's disease. NAD+ metabolism is central to the maintenance of synaptic structure and function. Its synthesis is mediated by nicotinamide mononucleotide adenylyltransferases (NMNATs), but their role in Parkinson's disease is not known. Here we report significantly decreased levels of NMNAT3 protein in the caudate nucleus of patients who have died with Parkinson's disease, which inversely correlated with the amount of monomeric α-synuclein. The detected alterations were specific and significant as the expression levels of NMNAT1, NMNAT2 and sterile alpha and TIR motif containing 1 (SARM1) were not significantly different in Parkinson's disease patients compared to controls. To test the functional significance of these findings, we ectopically expressed wild-type α-synuclein in retinoic acid-differentiated dopaminergic SH-SY5Y cells that resulted in decreased levels of NMNAT3 protein plus a neurite pathology, which could be rescued by FK866, an inhibitor of nicotinamide phosphoribosyltransferase that acts as a key enzyme in the regulation of NAD+ synthesis. Our results establish, for the first time, NMNAT3 alterations in Parkinson's disease and demonstrate in human cells that this phenotype together with neurite pathology is causally related to α-synucleinopathy. These findings identify alterations in the NAD+ biosynthetic pathway as a pathogenic mechanism underlying α-synuclein-mediated synaptopathy.

Nicotinamide N-Methyltransferase: An Emerging Protagonist in Cancer Macro(r)evolution.

Nicotinamide N-methyltransferase (NNMT) has progressed from being considered merely a Phase II metabolic enzyme to one with a central role in cell function and energy metabolism. Over the last three decades, a significant body of evidence has accumulated which clearly demonstrates a central role for NNMT in cancer survival, metastasis, and drug resistance. In this review, we discuss the evidence supporting a role for NNMT in the progression of the cancer phenotype and how it achieves this by driving the activity of pro-oncogenic NAD+-consuming enzymes. We also describe how increased NNMT activity supports the Warburg effect and how it promotes oncogenic changes in gene expression. We discuss the regulation of NNMT activity in cancer cells by both post-translational modification of the enzyme and transcription factor binding to the NNMT gene, and describe for the first time three long non-coding RNAs which may play a role in the regulation of NNMT transcription. We complete the review by discussing the development of novel anti-cancer therapeutics which target NNMT and provide insight into how NNMT-based therapies may be best employed clinically.

Potent Inhibition of Nicotinamide N-Methyltransferase by Alkene-Linked Bisubstrate Mimics Bearing Electron Deficient Aromatics.

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.

High Expression of Nicotinamide N-Methyltransferase in Patients with Sporadic Alzheimer's Disease.

We have previously shown that the expression of nicotinamide N-methyltransferase (NNMT) is significantly increased in the brains of patients who have died of Parkinson's disease (PD). In this study, we have compared the expression of NNMT in post-mortem medial temporal lobe, hippocampus and cerebellum of 10 Alzheimer's disease (AD) and 9 non-disease control subjects using a combination of quantitative Western blotting, immunohistochemistry and dual-label confocal microscopy coupled with quantitative analysis of colocalisation. NNMT was detected as a single protein of 29 kDa in both AD and non-disease control brains, which was significantly increased in AD medial temporal lobe compared to non-disease controls (7.5-fold, P < 0.026). There was no significant difference in expression in the cerebellum (P = 0.91). NNMT expression in AD medial temporal lobe and hippocampus was present in cholinergic neurones with no glial localisation. Cell-type expression was identical in both non-disease control and AD tissues. These results are the first to show, in a proof-of-concept study using a small patient cohort, that NNMT protein expression is increased in the AD brain and is present in neurones which degenerate in AD. These results suggest that the elevation of NNMT may be a common feature of many neurodegenerative diseases. Confirmation of this overexpression using a larger AD patient cohort will drive the future development of NNMT-targetting therapeutics which may slow or stop the disease pathogenesis, in contrast to current therapies which solely address AD symptoms.

Metabolic rewiring in melanoma drug-resistant cells.

Several evidences indicate that melanoma, one of the deadliest types of cancer, presents the ability to transiently shift its phenotype under treatment or microenvironmental pressure to an invasive and treatment-resistant phenotype, which is characterized by cells with slow division cycle (also called slow-cycling cells) and high-OXPHOS metabolism. Many cellular marks have been proposed to track this phenotype, such as the expression levels of the master regulator of melanocyte differentiation (MITF) and the epigenetic factor JARID1B. It seems that the slow-cycling phenotype does not necessarily present a single gene expression signature. However, many lines of evidence lead to a common metabolic rewiring process in resistant cells that activates mitochondrial metabolism and changes the mitochondrial network morphology. Here, we propose that mitochondria-targeted drugs could increase not only the efficiency of target therapy, bypassing the dynamics between fast-cycling and slow-cycling, but also the sensitivity to immunotherapy by modulation of the melanoma microenvironment.

Nicotinamide N-Methyltransferase: Genomic Connection to Disease.

Single-nucleotide polymorphisms (SNPs) in and around the nicotinamide N-methyltransferase (NNMT) gene are associated with a range of cancers and other diseases and conditions. The data on these associations have been assembled, and their strength discussed. There is no evidence that the presence of either the major or minor base in any SNP affects the expression of nicotinamide N-methyltransferase. Nevertheless, suggestions have been put forward that some of these SNPs do affect NNMT expression and thus homocysteine metabolism. An alternative idea involving non-coding messenger RNAs (mRNAs) is suggested as a possible mechanism whereby health is influenced. It is postulated that these long, non-coding NNMT mRNAs may exert deleterious effects by interfering with the expression of other genes. Neither hypothesis, however, has experimental proof, and further work is necessary to elucidate NNMT genetic interactions.

Nicotinamide N-methyltransferase expression in SH-SY5Y human neuroblastoma cells decreases oxidative stress.

Nicotinamide N-methyltransferase (NNMT) plays a central role in cellular metabolism, regulating pathways including epigenetic regulation, cell signalling, and energy production. Our previous studies have shown that the expression of NNMT in the human neuroblastoma cell line SH-SY5Y increased complex I activity and subsequent ATP synthesis. This increase in ATP synthesis was lower than the increase in complex I activity, suggesting uncoupling of the mitochondrial respiratory chain. We, therefore, hypothesised that pathways that reduce oxidative stress are also increased in NNMT-expressing SH-Y5Y cells. The expression of uncoupling protein-2 messenger RNA and protein were significantly increased in NNMT-expressing cells (57% ± 5.2% and 20.1% ± 1.5%, respectively; P = .001 for both). Total GSH (22 ± 0.3 vs 35.6 ± 1.1 nmol/mg protein), free GSH (21.9 ± 0.2 vs 33.5 ± 1 nmol/mg protein), and GSSG (0.6 ± 0.02 vs 1 ± 0.05 nmol/mg protein; P = .001 for all) concentrations were significantly increased in NNMT-expressing cells, whereas the GSH:GSSG ratio was decreased (39.4 ± 1.8 vs 32.3 ± 2.5; P = .02). Finally, reactive oxygen species (ROS) content was decreased in NNMT-expressing cells (0.3 ± 0.08 vs 0.12 ± 0.03; P = .039), as was the concentration of 8-isoprostane F2α (200 ± 11.5 vs 45 ± 2.6 pg/mg protein; P = .0012). Taken together, these results suggest that NNMT expression reduced ROS generation and subsequent lipid peroxidation by uncoupling the mitochondrial membrane potential and increasing GSH buffering capacity, most likely to compensate for increased complex I activity and ATP production.

Cholinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In vitro Model for Alzheimer's Disease Studies.

Cholinergic transmission is critical to high-order brain functions such as memory, learning, and attention. Alzheimer's disease (AD) is characterized by cognitive decline associated with a specific degeneration of cholinergic neurons. No effective treatment to prevent or reverse the symptoms is known. Part of this might be due to the lack of in vitro models that effectively mimic the relevant features of AD. Here, we describe the characterization of an AD in vitro model using the SH-SY5Y cell line. Exponentially growing cells were maintained in DMEM/F12 medium and differentiation was triggered by the combination of retinoic acid (RA) and BDNF. Both acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) enzymatic activities and immunocontent were determined. For mimicking tau and amyloid-ß pathology, RA + BDNF-differentiated cells were challenged with okadaic acid (OA) or soluble oligomers of amyloid-ß (AßOs) and neurotoxicity was evaluated. RA + BDNF-induced differentiation resulted in remarkable neuronal morphology alterations characterized by increased neurite density. Enhanced expression and enzymatic activities of cholinergic markers were observed compared to RA-differentiation only. Combination of sublethal doses of AßOs and OA resulted in decreased neurite densities, an in vitro marker of synaptopathy. Challenging RA + BDNF-differentiated SH-SY5Y cells with the combination of sublethal doses of OA and AßO, without causing considerable decrease of cell viability, provides an in vitro model which mimics the early-stage pathophysiology of cholinergic neurons affected by AD.

A feedback loop between dipeptide-repeat protein, TDP-43 and karyopherin-α mediates C9orf72-related neurodegeneration.

Accumulation and aggregation of TDP-43 is a major pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 inclusions also characterize patients with GGGGCC (G4C2) hexanucleotide repeat expansion in C9orf72 that causes the most common genetic form of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Functional studies in cell and animal models have identified pathogenic mechanisms including repeat-induced RNA toxicity and accumulation of G4C2-derived dipeptide-repeat proteins. The role of TDP-43 dysfunction in C9ALS/FTD, however, remains elusive. We found G4C2-derived dipeptide-repeat protein but not G4C2-RNA accumulation caused TDP-43 proteinopathy that triggered onset and progression of disease in Drosophila models of C9ALS/FTD. Timing and extent of TDP-43 dysfunction was dependent on levels and identity of dipeptide-repeat proteins produced, with poly-GR causing early and poly-GA/poly-GP causing late onset of disease. Accumulating cytosolic, but not insoluble aggregated TDP-43 caused karyopherin-α2/4 (KPNA2/4) pathology, increased levels of dipeptide-repeat proteins and enhanced G4C2-related toxicity. Comparable KPNA4 pathology was observed in both sporadic frontotemporal dementia and C9ALS/FTD patient brains characterized by its nuclear depletion and cytosolic accumulation, irrespective of TDP-43 or dipeptide-repeat protein aggregates. These findings identify a vicious feedback cycle for dipeptide-repeat protein-mediated TDP-43 and subsequent KPNA pathology, which becomes self-sufficient of the initiating trigger and causes C9-related neurodegeneration.

Cannabinoid-Based Therapies and Brain Development: Potential Harmful Effect of Early Modulation of the Endocannabinoid System.

The endocannabinoid retrograde signaling pathway is widely expressed in the central nervous system, where it plays major roles in regulating synaptic plasticity (excitatory and inhibitory) through long-term potentiation and long-term depression. The endocannabinoid system (ECS) components-cannabinoid receptors, endocannabinoids and synthesis/degradation enzymes-are expressed and are functional from early developmental stages and throughout adolescent cortical development, regulating progenitor cell fate, neural differentiation, migration and survival. This may potentially confer increased vulnerability to adverse outcomes from early cannabinoid exposure. Cannabidiol (CBD) is one of the most studied exogenous cannabinoids, and CBD-enriched Cannabis extracts have been widely (and successfully) used as adjuvants to treat children with refractory epilepsy, and there is even a Food and Drug Administration (FDA)-approved drug with purified CBD derived from Cannabis. However, there is insufficient information on possible long-term changes in the central nervous system caused by cannabinoid treatments during early childhood. Like the majority of cannabinoids, CBD is able to exert its effects directly and indirectly through the ECS, which can perturb the regulatory processes mediated by this system. In addition, CBD has a large number of non-endocannabinoid targets, which can explain CBD's effects. Here, we review the current knowledge about CBD-based therapies-pure and CBD-enriched Cannabis extracts-in studies with pediatric patients, their side effects, and their mechanisms of action regarding the central nervous system and neurodevelopment aspects. Since Cannabis extracts contain Δ9-tetrahydrocannabinol (Δ9-THC), we consider that pure CBD is possibly safer for young patients. Nevertheless, CBD, as well as other natural and/or synthetic cannabinoids, should be studied in more detail as a therapeutic alternative to CBD-enriched Cannabis extracts for young patients.

The kinetic analysis of the N-methylation of 4-phenylpyridine by nicotinamide N-methyltransferase: Evidence for a novel mechanism of substrate inhibition.

The N-methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N-methylation by nicotinamide N-methyltransferase (NNMT) and its effect upon 4-phenylpyridine toxicity in vitro. Human recombinant NNMT possessed 4-phenylpyridine N-methyltransferase activity, with a specific activity of 1.7 ±â€¯0.03 nmol MPP+ produced/h/mg NNMT. Although the Km for 4-phenylpyridine was similar to that reported for nicotinamide, its kcat of 9.3 × 10-5 ±â€¯2 × 10-5 s-1 and specificity constant, kcat/Km, of 0.8 ±â€¯0.8 s-1 M-1 were less than 0.15% of the respective values for nicotinamide, demonstrating that 4-phenylpyridine is a poor substrate for NNMT. At low (<2.5 mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a Ki of 34 ±â€¯8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a Ki of 4 ±â€¯1 mM. In silico molecular docking suggested that 4-phenylpyridine binds to the active site of NNMT in two non-redundant poses, one a substrate binding mode and the other an inhibitory mode. Finally, the expression of NNMT in the SH-SY5Y cell-line had no effect cell death, viability, ATP content or mitochondrial membrane potential. These data demonstrate that 4-phenylpyridine N-methylation by NNMT is unlikely to serve as a source of MPP+. The possibility for competitive inhibition by dimethylsulphoxide should be considered in NNMT-based drug discovery studies. The potential for 4-phenylpyridine to bind to the active site in two binding orientations using the same active site residues is a novel mechanism of substrate inhibition.

Inhibitors of nicotinamide N-methyltransferase designed to mimic the methylation reaction transition state.

Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyses the methylation of nicotinamide to form N'-methylnicotinamide. Both NNMT and its methylated product have recently been linked to a variety of diseases, suggesting a role for the enzyme as a therapeutic target beyond its previously ascribed metabolic function in detoxification. We here describe the systematic development of NNMT inhibitors derived from the structures of the substrates involved in the methylation reaction. By covalently linking fragments of the NNMT substrates a diverse library of bisubstrate-like compounds was prepared. The ability of these compounds to inhibit NNMT was evaluated providing valuable insights into the structural tolerances of the enzyme active site. These studies led to the identification of new NNMT inhibitors that mimic the transition state of the methylation reaction and inhibit the enzyme with activity on par with established methyltransferase inhibitors.

Mimicking Parkinson's Disease in a Dish: Merits and Pitfalls of the Most Commonly used Dopaminergic In Vitro Models.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and has both unknown etiology and non-curative therapeutic options. Patients begin to present the classic motor symptoms of PD-tremor at rest, bradykinesia and rigidity-once 50-70% of the dopaminergic neurons of the nigrostriatal pathway have degenerated. As a consequence of this, it is difficult to investigate the early-stage events of disease pathogenesis. In vitro experimental models are used extensively in PD research because they present a controlled environment that enables the direct investigation of the early molecular mechanisms that are potentially involved with dopaminergic degeneration, as well as for the screening of potential therapeutic drugs. However, the establishment of PD in vitro models is a controversial issue for neuroscience research not only because it is challenging to mimic, in isolated cell systems, the physiological neuronal environment, but also the pathophysiological conditions experienced by human dopaminergic cells in vivo during the progression of the disease. Since no previous work has attempted to systematically review the literature regarding the establishment of an optimal in vitro model, and/or the features presented by available models used in the PD field, this review aims to summarize the merits and limitations of the most widely used dopaminergic in vitro models in PD research, which may help the PD researcher to choose the most appropriate model for studies directed at the elucidation of the early-stage molecular events underlying PD onset and progression.

RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

Nicotinamide N-Methyltransferase in Health and Cancer.

Over the past decade, the roles of nicotinamide N-methyltransferase and its product 1-methyl nicotinamide have emerged from playing merely minor roles in phase 2 xenobiotic metabolism as actors in some of the most important scenes of human life. In this review, the structures of the gene, messenger RNA, and protein are discussed, together with the role of the enzyme in many of the common cancers that afflict people today.

A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine nitrogen of nicotinamide to generate N-methylnicotinamide. Interestingly, NNMT is also able to N-methylate a variety of other pyridine-containing small molecules, suggesting a secondary role for the enzyme in the detoxification of xenobiotics. A number of recent studies have also revealed links between NNMT overexpression and a variety of diseases, including multiple cancers, Parkinson's disease, diabetes, and obesity. To facilitate further study of both the substrate scope and potential for inhibitor development, we here describe the development of a new NNMT activity assay. The assay makes use of ultra-high-performance hydrophilic interaction chromatography, allowing for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection, providing for enhanced sensitivity and enabling high-throughput sample analysis. We successfully demonstrated the general applicability of the method by performing kinetic analyses of NNMT-mediated methylation for a range of pyridine-based substrates. These findings also provide new insight into the diversity of substrate recognition by NNMT in a quantitative manner. In addition, we further established the suitability of the assay for the identification and characterization of small molecule inhibitors of NNMT. To do so, we investigated the inhibition of NNMT by the nonspecific methyltransferase inhibitors sinefungin and S-adenosyl-l-homocysteine, revealing IC50 values in the low micromolar range. The results of these inhibition studies are particularly noteworthy as they will permit future efforts toward the development of new NNMT-specific inhibitors.

Nicotinamide N-methyltransferase catalyses the N-methylation of the endogenous ß-carboline norharman: evidence for a novel detoxification pathway.

Nicotinamide N-methyltransferase (NNMT) is responsible for the N-methylation of nicotinamide to 1-methylnicotinamide. Our recent studies have demonstrated that NNMT regulates cellular processes fundamental to the correct functioning and survival of the cell. It has been proposed that NNMT may possess ß-carboline (BC) N-methyltransferase activity, endogenously and exogenously produced pyridine-containing compounds which, when N-methylated, are potent inhibitors of Complex I and have been proposed to have a role in the pathogenesis of Parkinson's disease. We have investigated the ability of recombinant NNMT to N-methylate norharman (NH) to 2-N-methylnorharman (MeNH). In addition, we have investigated the toxicity of the BC NH, its precursor 1,2,3,4-tetrahydronorharman (THNH) and its N-methylated metabolite MeNH, using our in vitro SH-SY5Y NNMT expression model. Recombinant NNMT demonstrated NH 2N-methyltransferase activity, with a Km of 90 ± 20 µM, a kcat of 3 × 10(-4) ± 2 × 10(-5) s(-1) and a specificity constant (kcat/Km) of 3 ± 1 s(-1) M(-1) THNH was the least toxic of all three compounds investigated, whereas NH demonstrated the greatest, with no difference observed in terms of cell viability and cell death between NNMT-expressing and non-expressing cells. In NNMT-expressing cells, MeNH increased cell viability and cellular ATP concentration in a dose-dependent manner after 72 and 120 h incubation, an effect that was not observed after 24 h incubation or in non-NNNT-expressing cells at any time point. Taken together, these results suggest that NNMT may be a detoxification pathway for BCs such as NH.

The effect of foetal bovine serum supplementation upon the lactate dehydrogenase cytotoxicity assay: Important considerations for in vitro toxicity analysis.

The lactate dehydrogenase (LDH) assay is a commonly-used tool for assessing toxicity in vitro. However, anecdotal reports suggest that foetal bovine serum (FBS) may contain LDH at concentrations significant enough to interfere with the assay and thus reduce its sensitivity. A series of experiments were performed to determine whether addition of FBS to culture medium significantly elevated culture media LDH content, and whether replacement of FBS with heat inactivated foetal bovine serum (HI-FBS) reduced LDH content and interfered with cell response to cytotoxic challenge. The addition of FBS at 5, 10 and 15% final concentrations increased culture medium LDH content in a dose-dependent manner. The substitution of HI-FBS for FBS reduced culture medium LDH content and increased the dynamic range of the assay. Cell viability of the SH-SY5Y human neuroblastoma and N27 rat mesencephalic neurone cell lines were significantly reduced as measured using the MTT reduction assay, whilst HI-FBS only affected toxicity response in a cell- and toxin-specific manner, although these effects were small. Hence, for cell lines with a high FBS requirement, the use of HI-FBS or alternative toxicity assays can be considered, or the use of alternative formulations, such as chemically-defined serum-free media, be adopted.