Preface

Organisms living in the real world are inevitably exposed to many chemical, physical, and biological agents that are harmful: food additives, natural toxins, pesticides, nanomaterials, metals, radiation, and viruses, among others. However, most of these agents, if not all, may have unexpected consequences on the biota. Organisms are continuously exposed to heterogeneous xenobiotics released into different habitats either deliberately, inadvertently, or through non-regulated industrial discharges. Understanding how these agents can produce genetic alterations in DNA and what their role is in different biological systems continue to receive intense attention in fields such as health, pharmaceutical, environment, industry, agriculture, and food sectors.

Mutagenicity denotes the generation of stable changes in the DNA molecule that differ from the normal sequence of an organism, which may result in a transmissible change in the genotype of living organisms. Any damaged genetic material could result in mutations, thus stimulating carcinogenic progression or establishing a framework for hereditary disorders. Whereas mutations are generated mainly by exogenous agents, named mutagens, the term genotoxic describes the capability of those chemical, physical, and biological agents to directly affect the structure of DNA, the cellular spindle apparatus, and/or the topoisomerase enzymes that modulate DNA topology during DNA replication as well as chromosome segregation, which are, finally, responsible for the fidelity of the genome. However, genotoxic damage to DNA is not always associated with mutations. Spontaneous mutations arise from a variety of sources due to errors in DNA replication, repair, and recombination, and the presence of transposable genetic elements. Many agents can produce chemically reactive species during their metabolism, or are themselves reactive and may, therefore, cause irreversible changes to DNA.

Heritable changes are the origin of innate metabolic deficiencies in cellular systems, generating morbidity and mortality in organisms. Genetic disorders can be produced by a mutation in only one or in multiple genes, through a combination of gene mutations and environmental factors or by damage at the chromosomal level that affects the number and/or structure of entire chromosome(s), or parts thereof. Mutations in cells are not only involved in the initiation and promotion of several human diseases, including cancer, but are also implicated in several genetic disorders, like anaemia, diabetes, cardiovascular alterations, obesity, atherosclerosis, and numerous other degenerative disorders. Currently, scientists recognize more than 4,000 human diseases that are produced by mutations as a result of a combinatorial failure of more than one of these processes.

As indicated in a book we published some years ago, entitled "Genotoxicity-A Predictable Risk To Our Actual World", without knowledge of the mutagenic and genotoxic properties of chemical, physical, and biological agents, the evaluation of responses in living organisms, including humans, is difficult, and consequently the regulation of genotoxicants is a complex and difficult process. Accurate identification of the different classes of environmental genotoxicants and mutagens would permit international regulatory scientific agencies to use this information in a variety of legislative decisions to establish priorities of public and scientific concern.

We have attempted to compile information from different fields, highlighting the detrimental influence that mutagenic and genotoxic agents inflict on DNA and how antimutagenic and anticarcinogenic modulators are able to reduce the negative impact of such toxic agents on living species. Antimutagens and anticarcinogens are agents that decrease the number of mutations in cells, modulating host defence mechanisms. Therefore, knowledge regarding the mechanism of action of potentially mutagenic and/or carcinogenic agents provides the basis for elucidation of how these protective chemicals exert a response. Antimutagens are employed as one of the key methods to increase cellular resistance to mutagens. They are able to reduce or even remove the mutagenic effects exerted by toxic xenobiotics, stimulating compensatory repair and tolerance pathways in the DNA. In regard to their mode of action, antimutagens can act by influencing different targets, such as cellular membranes, DNA damage repair, replication, chromatin organization, and cell signalling.

This book opens with an interesting discussion about the use of yeast as a model organism for studying the biological effects of the P450-mediated metabolism of xenobiotics. This chapter also focuses on strategies for employing multiple genetic endpoints in screening chemicals, yeast strains that facilitate phenotyping cytochrome P450 polymorphisms to test the safety of thousands of chemicals, the limitations of animal systems, the advantages of model organisms, and the humanization of yeast cells by expressing human cytochrome P450 genes. The second chapter describes a possible molecular mechanism for how the addition of exogenous polyamines may increase the production of improved strains of filamentous fungi and the biotechnological applications of this phenomenon. The third chapter provides information on chemical and physical mutagenesis in breeding, exemplified by new modern homozygous self-pollinated sunflower lines, as well as additional recommendations on the use of methods to induce mutagenesis, including methods of generation, investigation, and subsequent use of mutations. The fourth chapter comprises an excellent review comparing the specific toxicity and genotoxicity exerted by heavy metals such as lead and cadmium using mammalian cells as a biological matrix in the context of ecotoxicology. The fifth chapter describes the importance of doublecortin-like kinase 1 (DCLK1), a member of the protein kinase superfamily and the doublecortin family, and its role in DNA damage response and repair, via direct and indirect mechanisms. It is well known that DCLK1 is expressed in cancer stem cells, and is implicated in initiating tumours. The sixth chapter reviews the role of oxidative stress induced by vanadium (a common mechanism of action of metal pollutants), observed in in vivo and in vitro systems, highlighting the way the production of free radicals inflicts damage in biomolecules including DNA, proteins, lipids, and carbohydrates. In addition, the chapter emphasizes the protective role of two water-soluble antioxidants, namely carnosine and ascorbate, present in biological systems. The seventh chapter constitutes an update on how the w-/w+ somatic mutation and recombination test of Drosophila melanogaster are employed extensively for antigenotoxicity analysis, focusing on actual published results to aid in the development of a reliable protocol in antigenotoxicity. Finally, this book comprises a chapter discussing the properties of antimutagenic substances with multiple mechanisms of action, in addition to introducing different aspects of natural and synthetic antimutagens.

**V**

Further, the chapter includes a brief compilation of scientific findings, either from dietary sources or synthetic agents, with potential to combat the disorders caused by the mutagenic agents, noting possible future perspectives and mechanisms of

The editors of Genotoxicity and Mutagenicity - Mechanisms and Test Methods are enormously grateful to all contributing authors for sharing their knowledge and insights in this book. They have made an extensive effort to gather the information included in every chapter. Readers are challenged to interpret the significance of various mechanisms and tested methodologies for detecting the causes and consequences of mutagenic and genotoxic agents. We hope that the topics discussed here encourage all those interested to explore new aspects of the fields of mutagenesis and genotoxicity by stimulating scientific dialogue. The publication of this book is of great importance to scientists, biologists, pharmacologists, physicians, and veterinarians, as well as engineers, teachers, graduate students, and administrators of environmental programmes, who can make use of these investigations to understand some aspects of mutagenic and genotoxic issues, making this volume a

**Sonia Soloneski Ph.D. and Marcelo L. Larramendy Ph.D.**

School of Natural Sciences and Museum,

National University of La Plata,

La Plata, Argentina

antimutagenics.

valuable reference in the future.

Further, the chapter includes a brief compilation of scientific findings, either from dietary sources or synthetic agents, with potential to combat the disorders caused by the mutagenic agents, noting possible future perspectives and mechanisms of antimutagenics.

The editors of Genotoxicity and Mutagenicity - Mechanisms and Test Methods are enormously grateful to all contributing authors for sharing their knowledge and insights in this book. They have made an extensive effort to gather the information included in every chapter. Readers are challenged to interpret the significance of various mechanisms and tested methodologies for detecting the causes and consequences of mutagenic and genotoxic agents. We hope that the topics discussed here encourage all those interested to explore new aspects of the fields of mutagenesis and genotoxicity by stimulating scientific dialogue. The publication of this book is of great importance to scientists, biologists, pharmacologists, physicians, and veterinarians, as well as engineers, teachers, graduate students, and administrators of environmental programmes, who can make use of these investigations to understand some aspects of mutagenic and genotoxic issues, making this volume a valuable reference in the future.

## **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**

Mutagens

*Michael Fasullo*

**Abstract**

damaging agents.

**1. Introduction**

recombination assays

Genotoxic Assays for Measuring

This review discusses using yeast as a model organism for studying the biological effects of P450-mediated metabolism of xenobiotics. We discuss the challenges of testing the safety of thousands of chemicals currently introduced into the market place, the limitations of the animal systems, the advantages of model organisms, and the humanization of the yeast cells by expressing human cytochrome P450 (CYP) genes. We discuss strategies in utilizing multiple genetic endpoints in screening chemicals and yeast strains that facilitate phenotyping CYP polymorphisms. In particular, we discuss yeast mutants that facilitate xenobiotic import and retention and particular DNA repair mutants that can facilitate in measuring genotoxic endpoints and elucidating genotoxic mechanisms. New directions in toxicogenetics suggest that particular DNA damaging agents may interact with chromatin and perturb gene silencing, which may also generate genetic instabilities. By introducing human CYP genes into yeast strains, new strategies can be explored for high-throughput testing of xenobiotics and identifying potent DNA

**Keywords:** cytochrome P450 polymorphisms, genotoxins, budding yeast,

Genotoxins are generally referred to as chemical agents that cause DNA damage,

which, in turn, can initiate recombination or mutation events or chromosome loss [1]. While mutagens and recombinagens are genotoxic, not all genotoxins are directly mutagenic [2]. Genotoxic exposure has been correlated to birth defects [3], cardiovascular disease [4], carcinogenesis [5], and accelerated aging [6]. Public health depends on minimizing exposure to genotoxic chemicals. Nonetheless, thousands of chemicals have yet to be tested, and new chemicals are annually synthesized. Federal agencies mandate that all chemicals be tested for safety before being introduced into the marketplace [7]. Generally, this testing has involved rapid screens for bacterial mutagenesis, micronuclei assays or comet assays for testing DNA fragmentation, and animal testing for determining carcinogenicity. Animal testing is often expensive and time-consuming and has increasingly raised ethical concerns. While microbial plate assays, such as the Ames test [8], have been standard in identifying chemical mutagens, some chemicals that test negative in

P450 Activation of Chemical

## **Chapter 1**
