Preface

Despite advances in science, a huge number of untested substances remain, necessitating that priorities are established for evaluating toxic inducers prior to their approval for animal and human uses. The toxicity of many xenobiotics is well-known today and interest in understanding the action mechanisms is being continuously stimulated by their continual re-evaluation. In this way, one of the major goals of cytotoxic estimation includes the preliminary screening of potential xenobiotics to determine the toxicity and efficacy of any substance, product or environmental agent, through employing *in vitro* approaches. These types of analyses are preferred in pilot testing in fields such as health, pharmaceuticals, environment, industry, agriculture and food, in order to estimate cell growth, reproduction and morphological effects.

Accurate adverse assessment by cytotoxic estimation as well as their role in different biological systems are a primary step employing for ranking the safety of many chemicals. In this regard, knowledge of relative toxicity is essential in order to determine a chemical's fate and thereby prevent or minimize after-effects. Similarly, the identification of cytotoxic responses may be essential for elucidating target cells and organ toxicity.

This book is intended to present some strategies, methods, interpretations and recent advances in order to facilitate scientific research on *in vitro* toxic responses, presenting both theoretical and practical aspects.

Although vast literature is available on cytotoxic evaluation, this book contains important investigations into the diverse chemical hazards encountered in anthropogenic and natural environments. Moreover, it provides valuable information regarding the toxicity of several xenobiotics that can negatively affect human beings. The contributors clearly discuss several concepts and approaches that will be useful for understanding the potential action mechanisms of various compounds, namely quinones, silver nanoparticles, antibiotics and plant extracts, among others.

This book is organized into six chapters. It begins with an introductory chapter that presents an overview of different approaches recommended for the preliminary *in vitro* screening of cellular response and analyses of dead cells within a cell population. The chapter highlights the strengths and weaknesses of some cytotoxic endpoints routinely employed and shows how the selection of an appropriate cytotoxicity bioassay is decisive in order to obtain a comprehensive toxicity profile. The second chapter presents a complete study highlighting the different responses of a cell to several xenobiotic agents as well as different tests that can be useful for evaluating cellular responses. The chapter focuses on the toxicological and cytotoxic evaluation of chemical substances through *in vitro* tests, as a competitive alternative to *in vivo* experimentation as a consequence of ethical considerations. The third chapter reviews the role of quinones – an important family of natural products mainly isolated from bacteria, fungi, algae, plants and other organisms – as possible agents to prevent cancer and microbial activity. The chapter summarizes the activities of 152 anticancer and 30 antimicrobial quinones, showing that these

categories of compounds exert an excellent response against numerous cell cancer lines and also exhibit good antimicrobial activity, determined by *in vitro* and *in vivo* studies. The fourth chapter discusses the impact of some methodological strategies in toxicity studies of silver nanoparticles on cultured cells. The chapter highlights the employment of detailed conditions required to correctly determine the nanoparticle size effect in studies of living cells and the invalidity problem of Smoluchowski's equation. It then describes an approach to analyze cytotoxicity when nanoparticles are stabilized with various surfactants. The fifth chapter provides toxic information on the assessment by lethal concentration 50 of a bacteriostatic antibiotic, namely sulfamethoxazole and its silver-sulfamethoxazole complex, employing brine shrimp as an experimental model. The final chapter discusses the medicinal properties of the plant *Evolvuluous alsinoides*, including multiple potential activities like antibiotic, antimicrobial, anti-inflammatory and antidiabetic properties. Furthermore, the chapter includes a brief compilation of scientific findings showing the protective role of phytochemicals under several pathological conditions.

The editors of *Cytotoxicity - New Insights into Toxic Assessment* are enormously grateful to all the contributing scientists for sharing their knowledge and insights in this interdisciplinary book project. They have made an extensive effort to arrange the information included in every chapter. The publication of this book is of high importance for students, researchers and scientists in diverse fields with expertise in toxicology, health, pharmaceuticals and other disciplines, who can contribute and share their findings to take this area forward for future investigations.

> **Sonia Soloneski Ph.D. and Marcelo L. Larramendy Ph.D.** School of Natural Sciences and Museum, National University of La Plata, La Plata, Argentina

> > **1**

**Chapter 1**

**1. Introduction**

Cytotoxicity

Introductory Chapter:

*Sonia Soloneski and Marcelo L. Larramendy*

biomolecules and medical devices, among others [1–5].

or at least to suggest a clue of the *in vivo* effects [7].

signaling or cell interaction, among other deleterious effects [8].

The evaluation of toxicity is an important process for assessing the hazards and risks that diverse xenobiotic released into the environment have for human beings, animals, plants and all compartments in our environment. In particular, the toxic response at cellular level plays a central role in the identification and quantification of adverse outcomes associated with exposure to numerous pollutants, including natural toxins, food additives, pesticides, nanomaterials, metals, radiation, viruses,

Conventional *in vivo* testing frequently requires a huge number of animal experiments. However, after application of Russel and Burch's strategy based on the '3Rs' – reduction, refinement and replacement – the replacement of animals of experimentation for research purposes has been considered, to minimize the cruelty and misuse that provoke pain, distress and death experiences [6]. Strategies employing specialized animal/human cell lines, tissue cultures, callus cultures, organ cultures and the continued use of long-established cultures are an excellent and practical way to screen the properties of any xenobiotic in the early stages of experimentation. It is well known that *in vitro* cytotoxicity cannot replace the conventional *in vivo* effects detected in the advanced stages of product development but cytotoxic estimation will assist in extrapolating *in vitro* observations to predict

Accurate assessment of the adverse effects of xenobiotics by estimating their cytotoxicity as well as their role in different biological systems is a primary step employed to rank the safety of many chemicals; knowledge of the relative toxicity is essential in order to decide the fate of a chemical to prevent or minimize their effects and to identify cytotoxic responses that may be essential for elucidating target cells as well as organ toxicity. According to Freshney [8], the meaning of cytotoxicity can differ, considering upon the nature of the study and whether cells are killed or simply have their metabolism altered. For example, whereas an anticancer drug employed in chemotherapy may be required to kill cells, the absence of toxicity in other chemicals may involve a deep complete analysis of specific targets such as modifications in cell

In recent years, science and technology innovations have accelerated the progress in the standardization of methods for determining cytotoxicity that are properly sensitive to predict several levels of cell toxicity, i.e., from low to high. These bioassays are efficient and economical tools that can quickly make valuable responses that are suitable for both qualitative and quantitative assessment [8].

There are numerous as well as highly recommended methodologies routinely used for preliminary *in vitro* screening of cellular response and the analysis of dead cells within a cell population. Advantages associated with *in vitro* approaches are that they are easy to follow, less time-consuming and less expensive than other developmental

## **Chapter 1**
