Control compounds for preclinical drug-induced liver injury assessment: Consensus-driven systematic review by the ProEuroDILI network.

BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, and herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. In this systematic review, we analyzed the compounds used in hepatotoxicity assays and established a list of DILI-positive and -negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from the COST Action ProEuroDILI Network (CA17112). METHODS: Following 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified 'Toxicological Data Reliability Assessment Tool'. RESULTS: A total of 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on drug concentrations. CONCLUSIONS: Extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative control drugs for validation of in vitro models of DILI is proposed. IMPACT AND IMPLICATIONS: Prediction of human toxicity early in the drug development process remains a major challenge, necessitating the development of more physiologically relevant liver models and careful selection of drug-induced liver injury (DILI)-positive and -negative control drugs to better predict the risk of DILI associated with new drug candidates. Thus, this systematic study has crucial implications for standardizing the validation of new in vitro models of DILI. By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining in vitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.

Decreasing Intracellular Entropy by Increasing Mitochondrial Efficiency and Reducing ROS Formation-The Effect on the Ageing Process and Age-Related Damage.

A hypothesis is presented to explain how the ageing process might be influenced by optimizing mitochondrial efficiency to reduce intracellular entropy. Research-based quantifications of entropy are scarce. Non-equilibrium metabolic reactions and compartmentalization were found to contribute most to lowering entropy in the cells. Like the cells, mitochondria are thermodynamically open systems exchanging matter and energy with their surroundings-the rest of the cell. Based on the calculations from cancer cells, glycolysis was reported to produce less entropy than mitochondrial oxidative phosphorylation. However, these estimations depended on the CO2 concentration so that at slightly increased CO2, it was oxidative phosphorylation that produced less entropy. Also, the thermodynamic efficiency of mitochondrial respiratory complexes varies depending on the respiratory state and oxidant/antioxidant balance. Therefore, in spite of long-standing theoretical and practical efforts, more measurements, also in isolated mitochondria, with intact and suboptimal respiration, are needed to resolve the issue. Entropy increases in ageing while mitochondrial efficiency of energy conversion, quality control, and turnover mechanisms deteriorate. Optimally functioning mitochondria are necessary to meet energy demands for cellular defence and repair processes to attenuate ageing. The intuitive approach of simply supplying more metabolic fuels (more nutrients) often has the opposite effect, namely a decrease in energy production in the case of nutrient overload. Excessive nutrient intake and obesity accelerate ageing, while calorie restriction without malnutrition can prolong life. Balanced nutrient intake adapted to needs/activity-based high ATP requirement increases mitochondrial respiratory efficiency and leads to multiple alterations in gene expression and metabolic adaptations. Therefore, rather than overfeeding, it is necessary to fine-tune energy production by optimizing mitochondrial function and reducing oxidative stress; the evidence is discussed in this paper.

The Central Role of the NAD+ Molecule in the Development of Aging and the Prevention of Chronic Age-Related Diseases: Strategies for NAD+ Modulation.

The molecule NAD+ is a coenzyme for enzymes catalyzing cellular redox reactions in several metabolic pathways, encompassing glycolysis, TCA cycle, and oxidative phosphorylation, and is a substrate for NAD+-dependent enzymes. In addition to a hydride and electron transfer in redox reactions, NAD+ is a substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases and even moderate decreases in its cellular concentrations modify signaling of NAD+-consuming enzymes. Age-related reduction in cellular NAD+ concentrations results in metabolic and aging-associated disorders, while the consequences of increased NAD+ production or decreased degradation seem beneficial. This article reviews the NAD+ molecule in the development of aging and the prevention of chronic age-related diseases and discusses the strategies of NAD+ modulation for healthy aging and longevity.

Antipsychotic Drug Aripiprazole Protects Liver Cells from Oxidative Stress.

Antipsychotics used to treat schizophrenia can cause drug-induced liver injury (DILI), according to the Roussel Uclaf Causality Assessment Method. The role of oxidative stress in triggering injury in these DILI cases is unknown. We repeatedly administrated two second-generation antipsychotics, aripiprazole and olanzapine, at laboratory alert levels to study underlying mechanisms in stress prevention upon acute oxidative stress. The drugs were administered continuously for up to 8 weeks. For this, hepatoma Fao cells, which are suitable for metabolic studies, were used, as the primary hepatocytes survive in the culture only for about 1 week. Four stress responses-the oxidative stress response, the DNA damage response and the unfolded protein responses in the endoplasmic reticulum and mitochondria-were examined in H2O2-treated cells by antioxidant enzyme activity measurements, gene expression and protein quantification. Oxidant conditions increased the activity of antioxidant enzymes and upregulated genes and proteins associated with oxidative stress response in aripiprazole-treated cells. While the genes associated with DNA damage response, Gadd45 and p21, were upregulated in both aripiprazole- and olanzapine-treated cells, only aripiprazole treatment was associated with upregulation in response to even more H2O2, which also coincided with better survival. Endoplasmic reticulum stress-induced Chop was also upregulated; however, neither endoplasmic reticulum nor mitochondrial unfolded protein response was activated. We conclude that only aripiprazole, but not olanzapine, protects liver cells against oxidative stress. This finding could be relevant for schizophrenia patients with high-oxidative-stress-risk lifestyles and needs to be validated in vivo.

Mechanistic aspects of carotenoid health benefits - where are we now?

Dietary intake and tissue levels of carotenoids have been associated with a reduced risk of several chronic diseases, including cardiovascular diseases, type 2 diabetes, obesity, brain-related diseases and some types of cancer. However, intervention trials with isolated carotenoid supplements have mostly failed to confirm the postulated health benefits. It has thereby been speculated that dosing, matrix and synergistic effects, as well as underlying health and the individual nutritional status plus genetic background do play a role. It appears that our knowledge on carotenoid-mediated health benefits may still be incomplete, as the underlying mechanisms of action are poorly understood in relation to human relevance. Antioxidant mechanisms - direct or via transcription factors such as NRF2 and NF-κB - and activation of nuclear hormone receptor pathways such as of RAR, RXR or also PPARs, via carotenoid metabolites, are the basic principles which we try to connect with carotenoid-transmitted health benefits as exemplified with described common diseases including obesity/diabetes and cancer. Depending on the targeted diseases, single or multiple mechanisms of actions may play a role. In this review and position paper, we try to highlight our present knowledge on carotenoid metabolism and mechanisms translatable into health benefits related to several chronic diseases.

Antioxidant Vitamins and Ageing.

The free radical theory of ageing (FRTA), presented by Denham Harman in 1950s, proposed that aerobic organisms age due to reactive oxygen species (ROS)/free radical induced damage that accumulates in cells over time. Since antioxidants can neutralize free radicals by electron donation, the most logical approach was to use them as supplements in order to prevent ageing. In this chapter, we will discuss the inability of antioxidant supplementation to improve health and longevity.Although many antioxidants are efficient free radical quenchers in vitro, their in vivo effects are less clear. Recent evidence from human trials implies that antioxidant supplements do not increase lifespan and can even increase the incidence of diseases. Synthetic antioxidants were unable to consistently prevent ROS-induced damage in vivo, possibly as dietary antioxidants may not act only as ROS scavengers. Antioxidants can have dichotomous roles on ROS production. They are easily oxidized and can act as oxidants to induce damage when present in large concentrations. In appropriate amounts, they can modulate cellular metabolism by induction of cell stress responses and/or activate cell damage repair and maintenance systems. Therefore, the antioxidants' beneficial role may be reversed/prevented by excessive amounts of antioxidant supplements. On the other hand, ROS are also involved in many important physiological processes in humans, such as induction of stress responses, pathogen defence, and systemic signalling. Thus, both "anti-oxidative or reductive stress" (the excess of antioxidants) as well as oxidative stress (the excess of ROS) can be damaging and contribute to the ageing processes.

Reduced risk of apoptosis: mechanisms of stress responses.

Apoptosis signaling pathways are integrated into a wider network of interconnected apoptotic and anti-apoptotic pathways that regulate a broad range of cell responses from cell death to growth, development and stress responses. An important trigger for anti- or pro-apoptotic cell responses are different forms of stress including hypoxia, energy deprivation, DNA damage or inflammation. Stress duration and intensity determine whether the cell's response will be improved cell survival, due to stress adaptation, or cell death by apoptosis, necrosis or autophagy. Although the interplay between enhanced stress tolerance and modulation of apoptosis triggering is not yet fully understood, there is a substantial body of experimental evidence demonstrating that apoptosis and anti-apoptosis signaling pathways can be manipulated to trigger or delay apoptosis in vitro or in vivo. Anti-apoptotic strategies cover a broad range of approaches. These interventions include mediators that prevent apoptosis (trophic factors and cytokines), apoptosis inhibition (caspase inhibition, stimulation of anti-apoptotic or inhibition of pro-apoptotic proteins and elimination of apoptotic stimulus), adaptive stress responses (induction of maintenance and repair, caspase inactivation) and cell-cell interactions (blocking engulfment and modified micro environment). There is a consensus that preclinical efficacy and safety evaluations of anti-apoptotic strategies should be performed with protocols that simulate as closely as possible the effects of aging, gender, risk factors, comorbidities and co-medications.

Unfolded Protein Response and Macroautophagy in Alzheimer's, Parkinson's and Prion Diseases.

Proteostasis are integrated biological pathways within cells that control synthesis, folding, trafficking and degradation of proteins. The absence of cell division makes brain proteostasis susceptible to age-related changes and neurodegeneration. Two key processes involved in sustaining normal brain proteostasis are the unfolded protein response and autophagy. Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases (PrDs) have different clinical manifestations of neurodegeneration, however, all share an accumulation of misfolded pathological proteins associated with perturbations in unfolded protein response and macroautophagy. While both the unfolded protein response and macroautophagy play an important role in the prevention and attenuation of AD and PD progression, only macroautophagy seems to play an important role in the development of PrDs. Macroautophagy and unfolded protein response can be modulated by pharmacological interventions. However, further research is necessary to better understand the regulatory pathways of both processes in health and neurodegeneration to be able to develop new therapeutic interventions.

Therapeutic Dosage of Antipsychotic Drug Aripiprazole Induces Persistent Mitochondrial Hyperpolarisation, Moderate Oxidative Stress in Liver Cells, and Haemolysis.

Aripiprazole has fewer metabolic side effects than other antipsychotics; however, there are some severe ones in the liver, leading to drug-induced liver injury. Repeated treatment with aripiprazole affects cell division. Since this process requires a lot of energy, we decided to investigate the impact of aripiprazole on rat liver cells and mitochondria as the main source of cellular energy production by measuring the mitochondrial membrane potential, respiration, adenosine triphosphate (ATP) production, oxidative stress, antioxidative response, and human blood haemolysis. Here, we report that mitochondrial hyperpolarisation from aripiprazole treatment is accompanied by higher reactive oxygen species (ROS) production and increased antioxidative response. Lower mitochondrial and increased glycolytic ATP synthesis demand more glucose through glycolysis for equal ATP production and may change the partition between the glycolysis and pentose phosphate pathway in the liver. The uniform low amounts of the haemolysis of erythrocytes in the presence of aripiprazole in 25 individuals indicate lower quantities of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH+H+), which is in accordance with a decreased activity of glucose 6-phosphate dehydrogenase and the lower dehydrogenase activity upon aripiprazole treatment. The lower activity of glucose 6-phosphate dehydrogenase supports a shift to glycolysis, thus rescuing the decreased mitochondrial ATP synthesis. The putative reduction in NADPH+H+ did not seem to affect the oxidised-to-reduced glutathione ratio, as it remained equal to that in the untreated cells. The effect of aripiprazole on glutathione reduction is likely through direct binding, thus reducing its total amount. As a consequence, the low haemolysis of human erythrocytes was observed. Aripiprazole causes moderate perturbations in metabolism, possibly with one defect rescuing the other. The result of the increased antioxidant enzyme activity upon treatment with aripiprazole is increased resilience to oxidative stress, which makes it an effective drug for schizophrenia in which oxidative stress is constantly present because of disease and treatment.

Adaptive response, evidence of cross-resistance and its potential clinical use.

Organisms and their cells are constantly exposed to environmental fluctuations. Among them are stressors, which can induce macromolecular damage that exceeds a set threshold, independent of the underlying cause. Stress responses are mechanisms used by organisms to adapt to and overcome stress stimuli. Different stressors or different intensities of stress trigger different cellular responses, namely induce cell repair mechanisms, induce cell responses that result in temporary adaptation to some stressors, induce autophagy or trigger cell death. Studies have reported life-prolonging effects of a wide variety of so-called stressors, such as oxidants, heat shock, some phytochemicals, ischemia, exercise and dietary energy restriction, hypergravity, etc. These stress responses, which result in enhanced defense and repair and even cross-resistance against multiple stressors, may have clinical use and will be discussed, while the emphasis will be on the effects/cross-effects of oxidants.

Current Uncertainties and Future Challenges Regarding NAD+ Boosting Strategies.

Precursors of nicotinamide adenine dinucleotide (NAD+), modulators of enzymes of the NAD+ biosynthesis pathways and inhibitors of NAD+ consuming enzymes, are the main boosters of NAD+. Increasing public awareness and interest in anti-ageing strategies and health-promoting lifestyles have grown the interest in the use of NAD+ boosters as dietary supplements, both in scientific circles and among the general population. Here, we discuss the current trends in NAD+ precursor usage as well as the uncertainties in dosage, timing, safety, and side effects. There are many unknowns regarding pharmacokinetics and pharmacodynamics, particularly bioavailability, metabolism, and tissue specificity of NAD+ boosters. Given the lack of long-term safety studies, there is a need for more clinical trials to determine the proper dose of NAD+ boosters and treatment duration for aging prevention and as disease therapy. Further research will also need to address the long-term consequences of increased NAD+ and the best approaches and combinations to increase NAD+ levels. The answers to the above questions will contribute to the more efficient and safer use of NAD+ boosters.

Preapoptotic cell stress response of primary hepatocytes.

UNLABELLED: Primary hepatocytes are an important in vitro model for studying metabolism in man. Caspase-9 and Bcl-2-associated X protein (Bax) are regulators of the apoptotic pathway. Here we report on the translocation of procaspase-9 and Bax from cytoplasm to nuclei as well as on dispersion of mitochondria; these processes occur after isolation of primary hepatocytes. The observed changes appear similar to those at the beginning of apoptosis; however, the isolated hepatocytes are not apoptotic for the following reasons: (1) cells have a normal morphology and function; (2) the mitochondria are energized; (3) there is no apoptosis unless it is induced by, e.g., staurosporine or nodularin. Staurosporine does not trigger apoptosis through activation of caspase-9, as its activity is detected later than that of caspase-3. We propose that the translocation of procaspase-9 and Bax into the nuclei reduces the ability to trigger apoptosis through the intrinsic apoptotic pathway. The shifts of procaspase-9 and Bax are reversible in the absence of the apoptotic trigger; the spontaneous reversion was confirmed experimentally for procaspase-9, whereas Bax shifted from the nuclei to the cytosol and mitochondria after the initiation of apoptosis. To distinguish this process from apoptosis, we call it preapoptotic cell stress response. It shares some features with apoptosis; however, it is reversible and apoptosis has to be induced in addition to this process. CONCLUSION: Knowledge on preapoptotic cell stress response is important for assessing the quality of the cells used in cell therapies, in regenerative medicine, and of those used for modeling metabolic processes.

DMSO modulates the pathway of apoptosis triggering.

We demonstrate here that distribution of caspase-9 influences the pathway of apoptosis triggering, since caspase-9 is activated efficiently only when it is distributed solely in the cytosol. Caspase-9 moves to the nuclei in a response to cell stress during isolation of primary hepatocytes; this is called preapoptotic cell stress response. The dimethyl sulfoxide (DMSO) treatment cannot prevent the migration of caspase-9 into the nuclei when it is added to primary hepatocytes immediately after isolation; however, it can trigger redistribution of caspase-9 from the nuclei into the cytosol when added 1 day post-isolation. This redistribution is temporary, since caspase-9 returns to the nuclei within 48 hours of DMSO treatment. Thereafter, some caspase-9 is retained in the nuclei of DMSO-treated hepatocytes for longer than in the nuclei of untreated hepatocytes. By measuring caspase activities, we demonstrate that the addition of DMSO to cell culture medium can temporarily normalize the susceptibility of hepatocytes for apoptosis triggering through the intrinsic pathway. DMSO contributes also to the prolonged pathway inactivation, i.e., by extending preapoptotic cell stress response. We propose that DMSO extends the survival of primary hepatocytes by modulating preapoptotic cell stress response, which could be exploited for extending the lifespan of other primary cell cultures.

Nature Versus Nurture: What Can be Learned from the Oldest-Old's Claims About Longevity?

Beneficial genetic or environmental factors that influence the length and quality of life can be evaluated while studying supercentenarians. The oldest-old can withstand serious/fatal illnesses more than their peers and/or their aging rate is decreased. Supercentenarians are an interesting group of individuals whose lifestyle is not particularly healthy according to the common guidelines, namely some of them seem to have similar harmful behaviors, but still manage to stay healthier for longer, and while eventually dying from the same degenerative diseases as the general population, they develop symptoms 20-30 years later. As there are not many supercentenarians by definition, it is worthwhile to diligently collect their data to enable future meta-analyses on larger samples; much can be learned from supercentenarians' habits and lifestyle choices about the aging process. Contributions of genetics, lifestyle choices, and epigenetics to their extended life span are discussed here.

Differential p16 expression levels in the liver, hepatocytes and hepatocellular cell lines.

BACKGROUND: One of the most frequently deleted genes in cancer is CDKN2A encoding p16. This protein is often overexpressed in senescent cells, while its suppression can bypass the oncogene-induced senescence to enable transformation and tumorigenesis. The roles of the protein p16 are recently being expanded from the cell cycle progression regulator to the cellular regulator interacting in several different pathways. Yet data on its liver and liver cells' expression are inconclusive. METHODS: The expression of the p16 gene in liver and liver cells was determined by RT-qPCR and compared to its protein amounts by western blotting. RESULTS: p16 is expressed at low levels in the liver and rat hepatocytes. Its expression varies from none to the considerable levels in the examined hepatocellular carcinoma cell lines (FaO and HepG2) and in immortalized mouse hepatocytes. Such significant expression differences of an important cellular regulator warrant the need to closely examine the differences in biochemical pathways correlated with the p16 expression when using hepatocytes and hepatoma liver models.

Antioxidants, Food Processing and Health.

The loss and/or modification of natural antioxidants during various food processing techniques and storage methods, like heat/thermal, UV, pulsed electric field treatment, drying, blanching and irradiation is well described. Antioxidants in their reduced form are modified mainly by oxidation, and less by pyrolysis and hydrolysis. Thus, they are chemically converted from the reduced to an oxidized form. Here we describe the neglected role of the oxidized forms of antioxidants produced during food processing and their effect on health. While natural antioxidants in their reduced forms have many well studied health-promoting characteristics, much less is known about the effects of their oxidized forms and other metabolites, which may have some health benefits as well. The oxidized forms of natural antioxidants affect cell signaling, the regulation of transcription factor activities and other determinants of gene expression. Very low doses may trigger hormesis, resulting in specific health benefits by the activation of damage repair processes and antioxidative defense systems. Functional studies determining the antioxidants' effects on the organisms are important, especially as reduced or oxidized antioxidants and their metabolites may have additional or synergistic effects.

From carotenoid intake to carotenoid blood and tissue concentrations - implications for dietary intake recommendations.

There is uncertainty regarding carotenoid intake recommendations, because positive and negative health effects have been found or are correlated with carotenoid intake and tissue levels (including blood, adipose tissue, and the macula), depending on the type of study (epidemiological vs intervention), the dose (physiological vs supraphysiological) and the matrix (foods vs supplements, isolated or used in combination). All these factors, combined with interindividual response variations (eg, depending on age, sex, disease state, genetic makeup), make the relationship between carotenoid intake and their blood/tissue concentrations often unclear and highly variable. Although blood total carotenoid concentrations <1000 nmol/L have been related to increased chronic disease risk, no dietary reference intakes (DRIs) exist. Although high total plasma/serum carotenoid concentrations of up to 7500 nmol/L are achievable after supplementation, a plateauing effect for higher doses and prolonged intake is apparent. In this review and position paper, the current knowledge on carotenoids in serum/plasma and tissues and their relationship to dietary intake and health status is summarized with the aim of proposing suggestions for a "normal," safe, and desirable range of concentrations that presumably are beneficial for health. Existing recommendations are likewise evaluated and practical dietary suggestions are included.

Healthy Lifestyle Recommendations: Do the Beneficial Effects Originate from NAD+ Amount at the Cellular Level?

In this review, we describe the role of oxidized forms of nicotinamide adenine dinucleotide (NAD+) as a molecule central to health benefits as the result from observing selected healthy lifestyle recommendations. Namely, NAD+ level can be regulated by lifestyle and nutrition approaches such as fasting, caloric restriction, sports activity, low glucose availability, and heat shocks. NAD+ is reduced with age at a cellular, tissue, and organismal level due to inflammation, defect in NAMPT-mediated NAD+ biosynthesis, and the PARP-mediated NAD+ depletion. This leads to a decrease in cellular energy production and DNA repair and modifies genomic signalling leading to an increased incidence of chronic diseases and ageing. By implementing healthy lifestyle approaches, endogenous intracellular NAD+ levels can be increased, which explains the molecular mechanisms underlying health benefits at the organismal level. Namely, adherence to here presented healthy lifestyle approaches is correlated with an extended life expectancy free of major chronic diseases.

Aripiprazole reduces liver cell division.

Effects of aripiprazole on dopamine regulation are being tested as a treatment for patients with a dual diagnosis of schizophrenia and addictions, often cocaine dependence. Aripiprazole has one of the fewest side-effects among the second-generation antipsychotics. Nevertheless, severe aripiprazole hepatotoxicity was reported in persons with a history of cocaine and alcohol abuse. Here we report that therapeutically relevant aripiprazole concentrations, equal to laboratory alert levels in patients' serum, reduce the rate of hepatocytes' division. This could be an underlying mechanism of severe liver injury development in the patients with a history of alcohol and cocaine abuse, the two hepatotoxic agents that require increased ability of liver self-regeneration. Monitoring liver functions is, therefore, important in the cases when aripiprazole is co-prescribed or used with drugs with potential hepatotoxic effects.

The potential use of natural products to negate hepatic, renal and neuronal toxicity induced by cancer therapeutics.

Different types of chemotherapeutics are used for cancer treatment. These drugs act on several signal pathways, lead to programmed cell death, and damage cancer cells. Although many specific mechanisms of action have been suggested for chemotherapeutics, there are still gaps in understanding their effects. They may affect different components of the cell, particularly proteins with specific functions, such as enzymes. Recently, targeted and immuno therapies were introduced for treatment of different cancers. However, many cancer patients still depend on traditional and well-known drugs. Doxorubicin and platinum-based drugs are among the most frequently used chemotherapeutics. They are highly cytotoxic for cancer cells, but they also act on healthy cells. Hence, it is crucial to understand the mechanisms involved in order to decrease their side effects. Natural products, many of which are also available over-the-counter, may be considered to decrease various cancer drug-induced side effects. This review focuses on the use of these compounds to overcome side effects of chemotherapeutics, primarily doxorubicin and cisplatin, in the liver, kidney, and neuronal systems.