Proteins are vital biological macromolecules that regulate various indispensible pathways and processes for cellular homeostasis. Misfolded and aggregated species of proteins target the protein homeostasis of the cell and are widely implicated in several neurological and metabolic disorders. Accumulation of such proteotoxic species instigates cellular dysfunction resulting in toxicity. The cellular response to proteotoxicity is regulated by a network of various proteins such as molecular chaperones, transport, and clearance machineries and the stress signaling pathways that regulates stability and functionality of the cell. Here, we discuss the mechanisms of molecular chaperones and degradation pathways that regulate the formation and accumulation of proteotoxic species in vitro and also emphasize on protein aggregation disorders involved in the progression of various diseases and mitigation of proteotoxicity.
Genetic materials form the basis of every cell and allow them to pass on the information from one generation to another. Any insult incurred by the cell leads to altered genetic material and thus damage to the intricate cell machinery and finally, cell death. Humans are constantly exposed to these insults due to exposure to ultraviolet rays or to carcinogens like tobacco smoke, food adulterants, and so on. Following the industrial revolution, there was an indiscriminate use of pesticides and other effluents, which continued to accumulate in the environment with advent of time. Humans are thus unknowingly exposed to such compounds, which are deleterious to their health. These compounds become integrated into the DNA and cause mutations and thus, DNA damage. Any chemical capable of rendering DNA damaged is, therefore, termed genotoxic. Over the past few decades, there have been immense advances in toxicological studies, which help us to identify various genotoxic agents. Whenever a cell is exposed to such genotoxic agents, the changes it introduces in the DNA can lead to carcinogenicity or pathogenesis of certain diseases like atherosclerosis or cardiac dysfunction.
The challenges of reducing and refining animal use in the assessment of safety and toxicity of agents that may pose hazard to humans and the environment are immense. Therefore, there is increasing interest and focus on in vitro methods for the reliable prediction of plausible human toxicity. Since in vitro systems are only partially representative of the situation in vivo, assays must be carefully designed and executed. Various national and international regulatory authorities are rapidly turning toward in vitro methods for the identification of hazards associated with chemical agents. With increasing encouragement from regulators, extensive efforts are underway at a global level to validate promising tests. The main focus of this chapter is the scientific and regulatory considerations for developing in vitro toxicity tests, their validation, and their acceptance by regulatory authorities concerned with authorizing the intended use of the chemical.
A gold standard for toxicity prediction in a living system is very much needed by the toxicology community. The enormous effort that gets unaccounted for between the numbers of successful drugs reaching the market and the investment during drug discovery needs to be addressed for better success. Classical methods are not sufficient enough anymore to predict the toxicity as a whole by taking into account the species specificity, drug metabolism, drug exposure, acute versus chronic effect, and existing ailment multiple effects occurring at the same time. Routine biochemical measurements typically include enzyme activity and metabolite concentration measurement in a cell-free environment. Observations gathered from one type of in vitro system remotely mimic the whole animal behavior. Moreover, these biochemical measurements are typically the end point of toxicity when the derangement in the biological processes causes terminal damage to the system. Onset as well as progress of toxicity can hardly be accounted for from these "end point"–type observations. A better predictive method need to be able to integrate various forms of alteration measured in multiple cell system under different condition—a steady accumulation of changes eventually culminates into an irreversible outcome. An endeavor to collate various results obtained from different in vitro systems on different measurements together may reveal the holistic picture behind toxicity.
Toxicology plays a pivotal role in driving global research and medicine. The complete understanding of this science is definitely needed for human welfare and cure. In vitro toxicology has given us a clearer understanding of the subject via the alternate strategies and has been instrumental in reducing animal-based approaches sufficiently. The field has progressed and evolved tremendously over the years so much so that today it is being viewed as the present and future of toxicology. Human-based in vitro models of toxicology are not only ethically sound but more fruitful in terms of results and data extrapolation. In the current chapter, we have introduced the subject of in vitro toxicology with an emphasis being on the past and current prevalent trends. The aim is to highlight the origin, establishment, and progress of the field along with an overview of how the in vitro strategies have benefited the field of toxicology at large.
The application of in vitro models in evaluating the toxicity of toxicants has enhanced our limited understanding of drugs and chemical-induced toxicity. From the public perspective, in vitro model systems are much more useful because their application does not involve live animals for toxicity testing. The advancement of the in vitro models for consistent, rapid, and cost-effective toxicity testing is of major importance. These in vitro model systems used in toxicity testing have diverse advantages together with the decrease in the number of animals, the reduced price of animal maintenance, as well as small amount of chemicals needed for testing, shortening of the time needed, and increase in throughput for evaluating large number of chemical compounds and their metabolism. One of the current major benefits of in vitro test system is their value for screening of chemicals, drugs, toxicants, pesticides, etc. Many different in vitro models have been in use over the years, in which cell lines are the best model for assessment of toxicity study of toxic compounds.
In vitro assays for toxicological studies is the refinement and replacement of animal testing in order to minimize the use of traditional animal-based toxicology studies. In the recent decades, in vitro methods have been routinely used as these can be correlated with the in vivo studies and can help in understanding a specific in vivo response in any given species. Optimization of cell culture is the major primary step to in vitro studies as it includes handling of cells and tissues outside the body under controlled conditions. Explant, organotype culture, primary, secondary cell lines, and three-dimensional culture methods are routinely used for in vitro screening of toxicants. On the basis of established in vivo toxicity assays, new in vitro toxicity test methods have been developed. In vitro cytotoxicity testing, biotransformation, and mechanistic investigations based studies can be effectively used as a cost-effective method for determination of toxicity.
The development of in vitro assays to assess chemical safety, as alternatives to animal testing, has become an important undertaking in toxicology research. Moreover, the research in this field has been moving more into the definition of adverse outcome pathways activated by chemical exposure, with the goal of relying less in apical endpoints for the assessment of chemicals' safety. Thus the need is not only for in vitro predictive toxicological assays, but also for these assays to provide a significant assessment of the biological activity associated with chemicals of interest, and with that, a better understanding of the underlying mechanisms of potential toxicity. This chapter is focused in the use of transcriptional profiling in in vitro systems to determine the potential estrogenic activity of chemicals of interest, as part of the assessment of chemical safety. This approach can be applied to address the potential effects of any given chemical in other hormonal systems (i.e., endocrine disruptors), as well as other modes of action.
The Draize eye test has been historically used to evaluate potential ocular effects of chemicals. However, the Draize eye test has been criticized in regard to its use of subjective evaluation criteria, physiological differences between test animals and humans, variability of scoring tissue effects, overprediction, and ethical concern regarding use of experimental animals. Several in vitro test methods have been developed to replace the Draize eye test. This chapter reviews (1) available in vitro test methods validated and adopted as Organization for Economic Co-operation and Development Test Guidelines, (2) challenges the currently available in vitro test methods, and (3) ongoing developments in the area of in vitro test methods for evaluation of ocular effects.
In vitro tests play an increasingly important role in the diagnosis, prophylaxis, and treatment of diseases and add immense value in the understanding of cellular and functional aspects of disease processes for better therapeutic interventions. In vitro studies have become an integral part of early drug development process. There has been progress in the development of in vitro test using test systems of variant levels (i.e., from whole cells to isolated enzymes) for toxicity evaluations. Due to the complexity of organisms of different species, it is evident that no single in vitro test can represent the entire spectrum of toxic potency of chemicals. This demonstrates the industry's commitment to the reduction, refinement, and replacement of whole-animal in vivo tests whenever possible. However, the in vitro environment in which the cell culture is grown and from the source these test systems and related material is derived from is also ideal for the proliferation of microorganisms. Any oversight on the safety precautions related to in vitro handling techniques and inability of a scientist to recognize the possible factors can be hazardous. While not selecting the appropriate material for the experiment not only can ruin the experiment but can also cause deleterious effects on the health of the scientist. The focus of this chapter is to provide an overview of fundamental safety issues associated with in vitro toxicity assays toward safety assessment and appropriate mitigating efforts to be considered for safety in in vitro laboratories.
Genetic toxicology testing is a weight-of-evidence approach to identify and characterize chemical substances that can cause genetic modifications in somatic and/or germ cells. Prediction of genetic toxicology using computational tools is gaining more attention and preferred by regulatory authorities as an alternate safety assessment for in vivo or in vitro approaches. Due to the cost and time associated with experimental genetic toxicity tests, it is essential to develop more robust in silico methods to predict chemical genetic toxicity. A number of in silico genotoxicity predictive tools/models are developed based on the experimental data gathered over the years. These in silico tools are divided into statistical quantitative structure-activity relationships (QSAR)-based approaches and expert-based systems. This chapter covers the state of the art in silico toxicology approaches and standardized protocols, essential for conducting genetic toxicity predictions of chemicals. This chapter also highlights various parameters for the validation of the prediction results obtained from QSAR models.
Benzene is an established leukemogen and hematotoxin in humans. However, the finding that benzene is a multiple-site carcinogen in rodents raises the possibility that other tissues could be susceptible to benzene-induced carcinogenicity, especially since a significant excess of squamous cell carcinomas and papillomas arise from epidermal and oral keratinocytes in benzene-exposed rats. Since inflammation and sustained hyperplasia are two integral components in carcinogenesis, the elaboration of proinflammatory cytokines and growth factors by keratinocytes might provide a mechanistic link between tumor initiation and promotion in benzene-induced cancers. We observed that the principal benzene metabolites, represented by hydroquinone, 1,4-benzoquinone, phenol, 1,2,4-benzenetriol, and catechol, significantly alters the production of transforming growth factor of (TGF)-α and interleukin (IL)-8 in human epidermal keratinocyte cultures. These cytokines represent the primary growth promoting factor and neutrophil chemotactant in the skin, respectively. Cytokine secretion correlated with the known redox potential of individual benzene metabolites and antioxidants, including dimethyl sulfoxide, 1,1,3,3-tetramethylthiourea, and N-acetylcysteine, attenuated the response. Binary combinations of selected benzene metabolites synergized in the induction of IL-8, while benzene, by itself, induced about a three-fold increase in IL-8 production. Taken together, our studies suggest that benzene and many of its phase I metabolites induce inflammatory cytokines and growth factors and this occurs through direct covalent binding or the generation of reactive oxygen species by autooxidation and reduction. The elaboration of proinflammatory cytokines and growth factors by keratinocytes in response to benzene and its principal metabolites may participate in benzene-induced skin carcinogenesis.
A panel of human bronchial epithelial (BEAS-2B) cell lines stably expressing one single CYP450 cDNA (CYP2A6, 2B6, 2D6, 2E1, 3A4, and 3A5) was developed by liposome-mediated transfection. The different cell lines showed high levels of CYP450 expression as determined by Western blot analysis. These genetically engineered cell lines were applied to evaluate the specificity of fluorescent substrates, such as 7-alkoxycoumarins, coumarins, and 7-alkroxyresorufins, in the analysis of human CYP450 catalytic activities. Additionally, the activation of procarcinogens (i.e., dimethylnitrosamine and aflatoxin B-1) to cytotoxic and genotoxic compounds was taken as reference to illustrate the potential of this system for pharmacotoxicologic applications.
Using udders from slaughtered cows, the percutaneous absorption of betamethasone-17,21-dipropionate was tested The organ was perfused with gassed tyrode solution for up to 6 h. A region of udder skin (100 cm(2)) was treated topically with betamethasone-17,21-dipropionate as an ingredient of solution, cream, and ointment (Diprosone) and as ingredient of gel and ointment (Diprosis, with propylene glycol as an additional ingredient). Betamethasone-17,21-dipropionate (Diprosone) was also administered on skin areas treated with acetone to disorganize the horny layer. The concentration of betamethasone-17,21-dipropionate was measured in perfusate fractions by high-performance liquid chromatography (HPLC).A maximum absorption rate of betamethasone-17,21-dipropionate was found after administration of the ointment with propylene glycol (Diprosis ointment). The treatment with acetone caused an increase of the absorption rate after application of betamethasone-17,21-dipropionate as ointment, while no increase was measurable after administration of the solution.In conclusion, the isolated perfused bovine udder is an in vitro model, which maintains bovine udder skin with an isolated vasculature in a viable state. Using this in vitro model, it is possible to compare the dermal penetration and absorption of substances after topical administration of different drug formulations.
This article reviews one of the applications of reconstructed skin or epidermis in pharmacotoxicology: their use in the in vitro evaluation of skin metabolism. The various skin models available nowadays are described. They include reconstructed human epidermis and reconstructed skin (with a epidermis and a viable dermis element). The results obtained with these models concerning basal and inducible monooxygenase activities and other metabolic activities on topically applied compounds are developed. Finally, the possible applications of these models in dermatology are considered, particularly the effect of imidazole derivatives on cytochrome P-450-dependent activities of human keratinocytes.
The use of cultured human hepatoma Rep G2 cells as a model to identify inducers of the phase II enzyme quinone reductase is examined. Cells were transiently transfected with a plasmid (pWNQO(1)tkCAT4.61kb) containing functional sequences in the 5'-flanking region of the human quinone reductase (NQO(1)) gene. In this plasmid, the nucleotides -587 to -379, containing a functional ARE, are linked to a CAT reporter. Benzylisothiocyanate, a known inducer of quinone reductase in animal models and in hepa1c1c7 cells, increased expression of the reporter gene after transient transfection into Rep G2 cells by up to 5.5-fold (at 1 mu M benzylisothiocyanate). In untransfected cells, this concentration of benzylisothiocyanate gave a small and not significant induction of quinone reductase activity (1.3-fold). The specific activity of quinone reductase in uninduced Rep G2 cells is 10-fold higher than found in the uninduced mouse hepatoma Repa1c1c7 cell line. These preliminary results suggest that transfection of pWNQO(1)tkCAT4.61kb into Rep G2 cells may be a suitable method to determine which compounds have the potential to induce QR transcription in humans.
We are currently validating the use of cytotoxicity tests for predicting the possible skin irritating potential of various topical formulations. The cytotoxicity is determined in fibroblast cultures using the neutral red release (NRR) assay and the MTT test, For most products, the MTT test is more sensitive and detects cytotoxic effects at lower concentrations than the NRR test.The results of the cytotoxicity tests show a good correlation with the skin irritation potential which we measure on hairless guinea pigs. In general, a product is unlikely to be irritating if the IC50 is higher than 250 mg/ml in the NRR test, or higher than 125 mg/ml in the MTT assay, after 5 min exposure. The overall correlation between the cytotoxicity test and the skin irritation potential is good (r(2) = 0.77 for 49 formulations tested).We found the cytotoxicity tests most useful as an aid for selecting the most promising among a series of similar formulations, thus significantly reducing the number of animal experiments. They can also be used for identifying the causative agent in an irritating complex mixture.
There is no doubt that retinoids have profound physiological and pharmacologic effects on epidermal differentiation and maintenance. However, the response of keratinocytes to retinoic acid (RA) is strikingly different whether they are cultured in vitro or whether epidermis is treated topically in vivo. In vitro, RA is able to inhibit completely the appearance of several epidermal-specific markers and to induce the ectopic appearance of several markers of nonkeratinized stratified epithelia, suggesting that a metaplasia has occurred. On the contrary, RA applied in vivo does not inhibit keratinization but provokes an increase of several markers of epidermal differentiation, white still inducing the expression of markers of nonkeratinized epithelia The work presented. in this paper shows that these different behaviors of the keratinocytes in vivo and in vitro are genuine, and not due to the fact that in vitro retinoids were added to the culture medium (''systemically''), before keratinization of the cultures, and in the presence of fetal calf serum and dermal components, while in vivo they were applied topically on the stratum corneum. We show that the ''in vitro behavior'' persists when retinoids are applied topically on the stratum corneum of keratinocyte cultures performed in defined medium on inert filters, suggesting that there are unexplored crucial regulatory pathways involved in the cutaneous response to retinoids in vivo.
The isolated perfused bovine udder skin (BUS) model was developed for studies concerning percutaneous absorption of pharmaceutical substances (Kietzmann et al., 1993). Additionally this in vitro method provides new possibilities for the assessment of the skin irritation potential. The comparison of the prostaglandin E-2-concentrations and MTT values of untreated. control sites obtained simultaneously to treated skirt sites showed the unimpaired vitality during the perfusion period of 20 individual bovine udder studies for assessment of skin irritation. Furthermore the results reveal no seasonal influences on the suitability of the organs perfused.Out of a number of tests concerning body care ingredients and formulations the results of two surfactants widely used were selected. Sodium lauryl sulfate (SLS) and alkyl polyglycoside (APG) were applied on the udder skin with low concentrations of 3% and 10% active substance, respectively, under occlusive conditions for 1 h and 5 h. The biological effects in the epidermal and dermal layers were characterized by means of prostaglandin E-2 assay and methyl tetrazolium salt dye conversion. The two surfactants could be clearly differentiated. The skin mildness of APG concerning cytotoxicity and synthesis of eicosanoids (e.g. irritancy after exposure up to 5 h) was proven in conformity with human studies.
The potential hepatotoxicity of a new drug is currently examined during its development. However, the fact that laboratory animals are not always predictive models for human Liver and that research with humans has important ethical limitations has led to a growing interest in in vitro methods for anticipating the potential hepatotoxicity of drugs in humans. Hepatic cellular models can be used in the early stages of drug development and, in the case of human hepatocytes, they can provide dit ect information about potential effects on human liver. The predictability of the data generated by in vitro models depends on the use of a cellular system that reproduces to a large extent the metabolic behavior of human liver, the choice of appropriate parameters for evaluating hepatotoxic effects in vitro, and correct designing of the experiments so that the results obtained in vitro will be relevant in vivo. With regard to the first point, primary culture of human hepatocytes is an excellent in vitro model as they retain the expression of almost all the typical liver functions in culture. They retain their characteristic in vivo drug-metabolizing activities, and most metabolites present in culture media are the same as those found in vivo. The strategy proposed here to investigate the potential hepatotoxicity of drugs includes the use of both cytotoxicity and cellular metabolism parameters. The former give information on the maximum drug concentration compatible with cell survival. The latter provide direct information about the extent to which cell metabolic functions are altered. Extrapolation of the in vitro experimental results to humans is the ultimate goal, but is difficult to achieve. Several factors are relevant in interpreting in vitro data in relation to the most probable effects in vivo: the sensitivity of the in vitro model used; the importance of the biochemical function affected and, in close connection with this, the reversibility of the effect; and the pharmacokinetics of the drug in vivo.
