Chemical Carcinogenesis PDF

CHEMICAL CARCINOGENESIS a s s o c. p ro f. d r. M a r t i n a G o b e c Faculty of pharmacy University of Ljubljana  GENETIC TOXICITY ▪ discovery of mutagenesis in the 1940s, notably by Hermann Muller's work on X-ray-induced mutations, m...

CHEMICAL CARCINOGENESIS a s s o c. p ro f. d r. M a r t i n a G o b e c Faculty of pharmacy University of Ljubljana  GENETIC TOXICITY ▪ discovery of mutagenesis in the 1940s, notably by Hermann Muller's work on X-ray-induced mutations, marked a significant milestone in recognizing the potential of agents to cause genetic damage ▪ genotoxicity refers to the ability of certain substances/factors to damage an organism's genetic material, particularly its DNA (this damage can lead to mutations, chromosomal alterations, or other harmful genetic effects) GENETIC RISK ▪ change in the DNA sequence of sex cells ▪ hereditary mutations ▪ oxidative DNA damages are the most common reason for germline mutations ▪ occurs in a single body cell of an individual after its conception ▪ acquired mutations ▪ most commonly caused environmental factors DNA  DNA DAMAGE DNA itself is not inherently chemically reactive under normal physiological conditions, however, certain environmental factors, exposure to chemicals, radiation, and metabolic processes can induce chemical changes in DNA since bases contain nucleophilic centers and unstable double bonds. If DNA damage occurs, cellular mechanisms like DNA repair, cell cycle checkpoint response, apoptosis or damage tolerance can be trigerred. CYTOTOXICITY GENOTOXICITY acute effect - cell death) a chronological effect - mutagenesis TYPES OF DNA CAUSES CHEMICAL ASSAYS DAMAGE CARCINOGENESIS mutations spontaneous cancer related genes in vitro chomosomal aberrations external types of cancerogens in vivo IARC classification  TYPES OF DNA DAMAGE mutation permanent alteration of the nucleotide sequence of the genome of an organism point mutations a change in a single nucleotide within the DNA sequence. Based on the nucleotide that changed, we can divide them into two groups: transition mutation one purine (adenine or guanine) is replaced by another purine, or one pyrimidine (cytosine or thymine) is replaced by another pyrimidine transversion mutation a purine is replaced by a pyrimidine or vice versa (e.g adenine is replaced by cytosine or thymine)  TYPES OF DNA DAMAGE point mutations BASED ON THE EFFECT Silent mutation Missense Mutation Nonsense mutation alters a nucleotide but does not change alters a single nucleotide, leading to the converts a codon that codes for an the amino acid sequence due to the substitution of one amino acid in the amino acid into a stop codon (e.g. TAA, redundancy of the genetic code resulting protein TAG or TGA), resulting in premature termination of protein synthesis conservative non-conservative  TYPES OF DNA DAMAGE point mutations EXAMPLE Sickle cell anemia Individuals with sickle cell anemia have a point mutation (adenine is replaced by thymine) in the haemoglobin gene. This nucleotide substitution results in a change in the amino acid sequence (glutamic acid → valine) and leads to the formation of hemoglobin S (HbS). The substitution causes a change in the overall structure of the hemoglobin protein, which tends to polymerize and form long, rigid structures. This polymerization leads to the characteristic sickle shape of red blood cells, reduced flexibility, and increased susceptibility to damage and premature destruction.  TYPES OF DNA DAMAGE mutation permanent alteration of the nucleotide sequence of the genome of an organism insertion and deletion involve the addition or removal of one or more nucleotides in the DNA sequence, potentially causing a shift in the reading frame and altering the entire amino acid sequence of the protein. divisible by three the reading frame of protein translation is preserved and these “in- frame” deletion/insertion mutations lead to a deletion or insertion of one or more amino acids within the encoded protein not divisible by three the reading frame of protein translation is disrupted → frameshift mutation, which alters the reading frame of the gene→ completely changes the amino acid sequence downstream of the mutation site → a severe type of mutation (most of the time it results in inactivation of the protein)  TYPES OF DNA DAMAGE insertion EXAMPLE Tay-Sachs disease In individuals with Tay-Sachs disease, there is an insertion of four nucleotides in the HEXA gene, which codes for the synthesis of the enzyme hexosaminidase A. This is essential for the breakdown of fatty molecules called GM2 ganglioside in nerve cells. The insertion disrupts the normal reading frame of the gene, leading to the synthesis of a nonfunctional and unstable form of Hex-A and subsequent accumulation of GM2 ganglioside in the nerve cells, which interferes with normal biological processes. TYPES OF DNA DAMAGE  TYPES OF DNA DAMAGE chromosomal aberrations structural abnormalities in the number or structure of chromosomes, double strand break is the principal lesion leading to the abnormality Karyotyping … is a laboratory technique that involves the visualization and analysis of an individual's chromosomes to assess their number, size, shape, and structural abnormalities. cells are arrested at the metaphase of cell division, since then the chromosomes are most condensed and visible. The DNA is stained, usually Giemsa stain, to create a banding pattern that enhances the visibility of individual chromosomes. This latter helps identifying and distinguishing chromosome aberrations. CLASTOGEN an agent that causes chromosomal breakage  TYPES OF DNA DAMAGE chromosomal aberrations numerical involve changes in the number of chromosomes in a cell, only a few numerical abnormalities support development to term Aneuploidy abnormal number of chromosomes (either as an extra chromosome or a missing one). For example: Down syndrome (trisomy 21), Turner syndrome (monosomy X), and Patau syndrome (trisomy 13). Polyploidy presence of extra sets of chromosomes, often incompatible with life  TYPES OF DNA DAMAGE chromosomal aberrations structural caused by breaks and rearrangements in chromosomes and aberrations affect the physical arrangement of chromosomal material divisible by three Structural aberrations often arise due to errors in cell division processes, such as non-disjunction, which can lead to deletions, duplications, or translocations. Exposure to certain environmental factors, including radiation and certain chemicals, can increase the risk of structural chromosomal aberrations. Structural chromosomal aberrations can lead to genetic disorders, developmental issues, intellectual disabilities and cancer, the effect depends on the specific chromosomes involved and the extent of the structural changes.  TYPES OF DNA DAMAGE chromosomal aberrations structural EXAMPLE Philadelphia chromosome The Philadelphia chromosome is a notable structural chromosomal aberration associated with hematological malignancies, particularly chronic myeloid leukemia (CML). It results from a reciprocal translocation between chromosomes 9 and 22, which leads to the formation of a fusion gen known as BCR-ABL1. Consequently an abnormal protein with constitutive tyrosine kinase activity is expressed, which leads to the overactivation of signaling pathways, promoting cell growth and inhibiting apoptosis. TYPES OF DNA DAMAGE POINT MUTATION sickle cell anaemia, phenylketonuria cancer of epithelial cells (Ras) STRUCTURAL ABERATIONS haemophilia, Duchene dystrophy lymphoma, leukemia (c-myc) NUMERIC ABERRATIONS Down‘s syndrom retinoblastoma, breast cancer CAUSES of DNA DAMAGE Internal - spontaneous External (exogenous) mage unavoidable; these DNA alterations likely make induced by the enviroment (e.g. exposure to radiation or significant contributions to the development of carcinogens, viruses) sporadic cancers and hereditary diseases (e.g. errors during DNA replication - especially during meiosis) radiation mismatches chemical agents deamination environmental toxins depurination and depyrimidination infectious agents oxidative damage  CAUSES of DNA DAMAGE Internal - spontaneous BASE MISMATCHES ▪ each base is chemically intact ▪ instead of forming the Watson – Crick base pairs (A : T, C : G), the base is incorrectly paired in the duplex DNA ▪ major source of DNA base mismatches is replication ▪ error rate of ∼ 10− 9 per base pair per replication (one typo per 1 million pages)  CAUSES of DNA DAMAGE Internal - spontaneous BASE MISMATCHES ▪ cytosine (C), adenine (A), and guanine (G) contain an exocyclic amino group, which can deaminate ▪ deamination of cytosine occurs at a much higher rate than that of adenine or guanine (C → T mutation). It is usually caused by random collision of a water molecule with the bond that links the amino group of the base to the pyrimidine or purine ring.  CAUSES of DNA DAMAGE Internal - spontaneous DEPURINATION AND DEPYRIMIDINATION ▪ the glycosidic bond links the deoxyribose and the base, however, it is labile under physiological conditions → susceptible to hydrolysis ▪ DNA in the human cell loses thousands of purine bases everyday.  CAUSES of DNA DAMAGE Internal - spontaneous OXIDATIVE DAMAGE ▪ inevitable consequence of cellular metabolism ▪ the major ROS in cells include superoxide radical, hydrogen peroxide, and hydroxyl radical. The hydroxyl radical is the main cause of spontaneous oxidative DNA damage in cells since it readily interacts with DNA bases: ✓ oxidative base damage → over 80 different lesions (e.g. 8 –oxogaunine, which is highly mutagenic, because it can base pair with either C or A and oftenly resulty in G → T transversion mutations) ✓ single-strand and double-strand breaks ✓ DNA-protein crosslinks  CAUSES of DNA DAMAGE External (exogenous) Our environment is filled with factors (e.g. UV-light, radiation, chemicals) which can directly damage DNA or generate reactive metabolites that cause DNA lesions. Understanding and mitigating these exposures are essential for maintaining genomic health. Genotoxicity assessment is an essential component of the safety assessment of all types of substances, ranging from pharmaceuticals, industrial chemicals, pesticides, biocides, food additives, cosmetics ingredients, to veterinary drugs, relevant in the context of international legislations aiming at the protection of human and animal health.  CAUSES of DNA DAMAGE Repair mechanisms  CHEMICAL CARCINOGENESIS CANCER CHEMICAL CARCINOGENESIS 9.6 X 106 death rate/year  CHEMICAL CARCINOGENESIS Causes ▪ genetic factors ▪ infectious agents ▪ environmental factors ▪ immune system dysfunction ▪ lifestyle factors  CHEMICAL CARCINOGENESIS CANCER ▪ characterized by the uncontrolled growth of an abnormal cell to produce a population of cells that have acquired the ability to multiply and invade surrounding and distant tissues ▪ a heterogenous disease ▪ incidence of most cancers increase exponentially with age ▪ three to seven critical mutations or “hits” within a single cell are required for cancer development  CHEMICAL CARCINOGENESIS CANCEROGENESIS ▪ INITIATION: induction of a mutation in proto-oncogenes and/or inactivation of tumour-suppressor genes. Initiation is a rapid, irreversible process triggered by chemical or physical agents (known as initiating agents or genotoxic agents). ▪ PROMOTION: under the influence of other endogenous or exogenous chemical compounds (growth stimuli) the initiated cells are subject to clonal growth. This is why these exogenous and endogenous compounds are called tumour promoters. They are not mutagenic by themselves but trigger other mechanisms, such as changes in gene expression that are continued in all subsequent daughter cells. Cell proliferation rate increases and apoptotic cell death rate decreases. Promotion is a reversible process and only works in initiated cells. Well known promoters are phenobarbital, benzene, asbestos and arsenic. ▪ PROGRESSION: involves additional genotoxic events (chromosomal aberrations and translocations). Progression is an irreversible process leading to the formation of neoplasms.  CHEMICAL CARCINOGENESIS CANCER & GENES ONCOGENES ▪ proto-oncogenes are highly conserved genes that are involved in the control of normal cellular proliferation, differentiation and apoptosis. If they are altered by a mutation they become oncogenes (over 100 identified) ▪ changes tha lead to formation of oncogenes ✓ gain-of-function mutations ✓ amplification ✓ chromosomal translocations ✓ aberrant expression  CHEMICAL CARCINOGENESIS CANCER & GENES ONCOGENES: Ras ▪ Ras proteins function as membrane-associated molecular switches. The off position is when it is bound to guanosine diphosphate (GDP). Stimulation by a growth factor receptor, Ras exchanges GTP guanosine triphosphate for GDP, and now Ras is in the on position → kinase cascade activation → activation of several transcription factors → ↑ cell proliferation ▪ mutated ras is stuck in the “on” position ▪ 20–30% of all human tumors contain mutated ras  CHEMICAL CARCINOGENESIS CANCER & GENES TUMOR SUPPRESSOR GENES ▪ encode proteins that generally function as negative regulators of cell growth or regulators of cell death, some function in DNA repair and cell adhesion. ▪ approximately 18 known tumor suppressor genes (e.g., p53, Rb, APC, p16, and BRCA1) ▪ mutated tumor suppressor genes are inactivated and are no longer capable of negatively regulating cellular growth leading to specific forms of cancer predisposition  CHEMICAL CARCINOGENESIS CANCER & GENES TUMOR SUPPRESSOR GENES: p53 ▪ p53 is a transcription factor and participates in many cellular functions, including cell cycle regulation, DNA repair, and apoptosis ▪ single missense mutations can inactivate the p53 ▪ most frequently known mutated gene in human cancer (in approximately 70% of colon cancers, 50% of breast and lung cancers, and 97% of primary melanomas) ▪ different carcinogens cause different characteristic mutations in p53  CHEMICAL CARCINOGENESIS Carcinogens are ubstances with carcinogenic properties. They can be physical agents such as ionizing radiation, chemicals like certain pollutants and tobacco smoke, biological agents including certain viruses and bacteria, and even specific lifestyle factors like certain dietary choices and behaviors. Carcinogens can exert their effects through different mechanisms. Some may directly damage DNA, while others act as promoters. GENOTOXIC CARCINOGENS directly interact with and damage DNA EPIGENETIC CARCINOGENS by changing its structure do not directly damage DNA (through covalent bonds) but affect its expression or make the cell more sensitive to other agents  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS About 80% of tumours in humans are triggered by exogenous chemical agents and are not necessarily associated with direct exposure to them (may also arise from normal metabolism, oxidative stress, or chronic inflammation). Chemical genotoxic carcinogens are divided into two main groups: ▪ DIRECT-ACTING CARCINOGENS cause cancer without metabolic activation or chemical modification (activation-independent), as they damage DNA from within. These chemicals are also known as parent compounds or ultimate carcinogens. The most common are epoxides, imines, and alkyl and sulphate esters. ▪ INDIRECT-ACTING CARCINOGENS become carcinogenic after metabolic activation. Typical indirect carcinogens are polycyclic aromatic hydrocarbons (PAHs, benzo[a]pyrene in particular), nitrosamines, nitrosoureas, and aromatic amines.  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS..may act directly or after biotransformation.  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS Almost all of the cellular DNA damage reactions fall into just two general categories: ▪ the reaction of a DNA nucleophile with an electrophile ▪ the reaction of a DNA pi bond or C–H bond with a radical  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS the reaction of a DNA nucleophile with an electrophile ▪ most vulnerable to adduct forming are the following base sites: N3, O2, and O4 of thymine, N1, N3, and N7 of adenine, N3 and O2 of cytosine, and N2, N7, and O6 of guanine ▪ the reactivity of purine bases is higher than of the pyrimidine bases (G>A>>C>T) ▪ most common reaction is alkylation (N7 and O6 site of guanine)  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS the reaction of a DNA nucleophile with an electrophile Benzo[a]pyrene Activation and Adduct Formation Benzo[a]pyrene is a polycyclic aromatic hydrocarbon that forms during the incomplete combustion of organic matter and if it is biologically activated by metabolism a highly reactive epoxide is formed, which acts as an electrophile and forms a bulky adduct preferentially with guanine residues in DNA. These cause a lesion in the DNA and if left unrepaired, during DNA replication an adenine will usually be placed across from the lesion in the daughter molecule. Subsequent repair of the adduct will result in the replacement of the damaged guanine base with a thymine, causing a G –> T transversion mutation.  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS the reaction of a DNA pi bond or C–H bond with a radical ▪ radicals can react with bases via hydrogen atom abstraction or by addition to the pi bonds in the heterocyclic nucleobases ▪ important chemical event in neoplasm formation is the hydroxylation of DNA bases as a result of interaction. between a hydroxyl radical (OH ) and base (e.g. 8-hydroxyguanine (8-OH-dG), thymine glycol) 8-OH-dG thymine glycol  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS the reaction of a DNA pi bond or C–H bond with a radical Asbestosis and lung cancer Asbestos is the name given to a group of naturally occurring fibrous minerals that are resistant to heat and corrosion. Because of these properties, asbestos has been used in commercial products such as insulation and fireproofing materials, automotive brakes, and wallboard materials. Prolonged exposure to asbestos has been linked to an increased risk of lung cancer and other respiratory diseases, in part, to the generation of reactive oxygen species (ROS) and free radicals during the interaction between asbestos fibers and cellular components.  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS & PHOTOEXCITATION Ultraviolet light, specifically UVA and UVB, is known to induce various types of DNA damage, including the formation of DNA adducts, strand breaks, and the generation of reactive oxygen species.  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS & PHOTOEXCITATION ▪ Photogenotoxicity refers to the ability of a substance to cause genetic damage when exposed to light. The chemicals, after photoexcitation, can: → affect DNA directly (e.g psoralens and phenothiazines) → excite the surrounding molecules (e.g. porphyrins and riboflavns) → damage DNA via ROS formation (e.g. furocumarin hydroperoxides). Whichever the photogenotoxic mechanism, the compound must be excited close to the target (DNA). FOR WHICH COMPOUNDS IS PHOTOGENOTOXICITY TESTING INDICATED?  CHEMICAL CARCINOGENESIS CHEMICAL CARCINOGENS & PHOTOEXCITATION PUVA therapy Psoralens are a group of naturally occurring or synthetic compounds, which can become toxic when exposed to ultraviolet (UV) light, since they become activated and can form covalent bonds with DNA, leading to the formation of DNA adducts and crosslinks. Psoralens are used in a treatment known as PUVA (psoralen plus ultraviolet A) therapy for certain skin conditions, such as psoriasis and vitiligo, since phototherapy is believed to induce programmed cell death in T lymphocytes.  CHEMICAL CARCINOGENESIS EPIGENETIC CARCINOGENS Epigenetic changes involve modifications to the structure of DNA or its associated proteins, influencing how genes are turned on or off. Unlike genetic mutations, epigenetic changes are reversible and can be influenced by various environmental factors, including exposure to certain chemicals or compounds.  CHEMICAL CARCINOGENESIS EPIGENETIC CARCINOGENS Promoters ✓ liver enzyme-inducer type hepatocarcinogens (e.g. DDT) ✓ urothelial cell proliferation enhancers (e.g. sodium saccharin) ✓ skin tumor enhancers (e.g. croton oil) Endocrine-modifiers ✓ antiandrogens ✓ estrogenic hormones Immunomodulators Carcinogens that are non-genotoxic, non-apoptotic, ✓ cyclosporine ✓ purine analogs (e.g. azathioprine) and non-cytotoxic to the cell bu can still contribute to Peroxisome proliferator-activated-receptor (PPAR) α/γ agonist ✓ hypolipidemic fibrates carcinogenesis in an epigenetic manner, by directly ✓ phthalates Inorganic compounds affecting gene expression during transcription, ✓ metal or metal salt ✓ fiber (eg. Asbestos) translation, and post-translational events.  CHEMICAL CARCINOGENESIS EPIGENETIC CARCINOGENS Diethylstilbestrol (DES) is a synthetic form of the female hormone estrogen. It was prescribed to pregnant women from 1940 to prevent miscarriage, premature labor. In 1971, researchers linked prenatal DES exposure to a type of cancer of the cervix and vagina called clear cell adenocarcinoma. DES is now known to be an endocrine-disrupting chemical and females exposed to DES in utero, commonly called DES daughters, are at increased risk of several specific cancers. DES affects the methylation patterns of genes that are associated with proliferation, apoptosis (and growth factors.  CHEMICAL CARCINOGENESIS Key characteristics of carcinogens CARCINOGENS ▪ Is electrophilic or can be metabolically activated to an electrophile ▪ Is genotoxic ▪ Alters DNA repair or causes genomic DNA-REACTIVE CARCINOGENS EPIGENETIC CARCINOGENS instability ▪ Induces epigenetic alterations DNA-reactive indirectly produce DNA alterations/damage ▪ Induces oxidative stress ✓ DNA adducts formation ✓ mitogens stimulate proliferation ▪ Induces chronic inflammation ✓ other types of direct DNA damage ✓ alter DNA repair or cause genomic instability ▪ Is immunosuppressive ▪ Modulates receptor-mediated effects potentially mutagenic or cytotoxic ✓ modulate receptor-mediated effects ▪ Causes immortalization ✓ alter cell proliferation, cell death ▪ Alters cell proliferation, cell death, or ✓ induce chronic inflammation or immunosuppression nutrient supply ✓ inhibit cell–cell communication ✓ induce oxidative stress ▪ most require metabolic activation to become reactive, some act ▪ shifts in sites of tumor induction by modifiers of biotransformation not as an electrophile directly reported ▪ neonates often more sensitive ▪ little evidence of enhanced susceptibility of neonates, except saccharin ▪ some exhibit transplacental carcinogenicity ▪ little evidence for transplacental carcinogenicity, except diethylstilbestrol and saccharin ▪ many are active at low dosage ▪ may be active at low dosage, but require a level and duration of exposure to produce relevant cellular effect ▪ represent human hazards ▪ human carcinogenicity seen with hormonal or immunosuppressive agents ▪ additivity of carcinogenicity possible ▪ additivity of carcinogenicity uncertain (some can inhibit one another)  CHEMICALASSAYS in vivo CARCINOGENESIS GENOTOXICITY & METABOLISM Cytochrome P450 enzymes are involved in the metabolism of a wide range of drugs and environmental chemicals, including some pro-carcinogens. However, individuals can have different variants (or alleles) of these genes, which can lead to varying levels of enzyme activity. These variants can be categorized into different phenotypes: ▪ Extensive Metabolizers (EMs): individuals with two fully functional copies of the genes are considered extensive metabolizers. They have normal enzyme activity and are able to metabolize substrates efficiently. ▪ Intermediate Metabolizers (IMs): have one fully functional copy and one non-functional or reduced-function copy of the genes. This results in reduced enzyme activity, which can lead to altered metabolism of certain substances. ▪ Poor Metabolizers (PMs): Poor metabolizers have two non-functional or severely reduced-function copies of the genes. As a result, they have significantly impaired enzyme activity, which can lead to slower or inadequate metabolism of certain substances. ▪ Ultra-rapid Metabolizers (UMs): Some individuals may have multiple copies of the functional genes, leading to higher-than-normal enzyme activity. This can result in rapid metabolism of substrates.  CHEMICAL CARCINOGENESIS GENOTOXICITY & METABOLISM Aflatoxin B1 and metabolism Aflatoxin B1 is a naturally occurring mycotoxin produced by certain molds and by itself it is not highly genotoxic. When ingested, it is metabolized by liver enzymes, particularly CYP1A5 and CYP3A3. It can be transformed into its more reactive and genotoxic form, aflatoxin B1-8,9-epoxide, which binds covalently to DNA, forming adducts → depurination → development of liver cancer. The ability of an individual to metabolize AFB1 can be influenced by genetic variations in cytochrome P450 enzymes: extensive metabolizers may be at a higher risk for genotoxic effects individuals with reduced function or non-functional may have a lower capacity to activate aflatoxin B1, potentially reducing their susceptibility to its genotoxic effects.  ASSAYS three main lists of internationally recognized carcinogens exist: those of IARC (International Agency for Research on Cancer), of the ACGIH (American Conference of Governmental Industrial Hygienists) and of the European Union (CLP) classified by the weight of evidence for carcinogenicity referred to as sufficient, limited, or inadequate based on both epidemiological studies and animal data  ASSAYS IARC CLASSIFICATION Group 1: Carcinogenic to Humans This category is used when there is sufficient evidence to conclude that the agent is carcinogenic to humans. The evidence may come from epidemiological studies, animal experiments, or other relevant data. Notable examples in Group 1 include tobacco smoke, asbestos, and certain chemicals. Group 2: Probably Carcinogenic to Humans This category is used when there is limited evidence of carcinogenicity in humans and sufficient evidence in experimental animals. There may be some uncertainties regarding the interpretation of the available data. Group 2 is subdivided into Group 2A (probably carcinogenic) and Group 2B (possibly carcinogenic). Examples in Group 2A include formaldehyde, and in Group 2B, glyphosate. Group 3: Unclassifiable as to Carcinogenicity in Humans This category is used when the available evidence is inadequate to determine the carcinogenicity of the agent in humans. The classification does not imply that the agent is non-carcinogenic, only that the evidence is not sufficient for a clear determination.  ASSAYS ASSESSMENT OF GENOTOXIC HAZARD Comprehensive safety assessments for chemicals used in various industries, including pharmacy, cosmetics, food, and agriculture include genotoxicity testing as a crucial component. A battery approach is reasonable because no single test is capable of detecting all genotoxic mechanisms relevant in tumorigenesis  ASSAYS ASSESSMENT OF GENOTOXIC HAZARD Guideline ICH S2(R1): genotoxicity testing and data interpretation for pharmaceuticals intended for human use OPTION 1 OPTION 2 1. In vitro Test for gene mutations in bacteria (Ames test) 1. In vitro Test for gene mutations in bacteria (Ames test) 2. In vitro est for chromosomal damage in mammalian cells 2. In vivo test for chromosomal damage (blood or bone ▪ in vitro chromosomal aberration assay or marrow): an in vivo assessment of genotoxicity with two ▪ in vitro micronucleus assay or different tissues, usually an assay for micronuclei using rodent ▪ in vitro mouse lymphoma TK gene mutation assay hematopoietic cells and a second in vivo assay. Typically this 3. In vivo test for chromosomal damage (generally a test for would be a DNA strand breakage comet assay in liver, unless chromosomal damage using rodent hematopoietic cells, otherwise justified. either for micronuclei or for chromosomal aberrations in metaphase cells) In vitro Test for gene mutations in bacteria (Ames test) ASSAYS In vitro est for chromosomal damage in mammalian cells in vitro in vitro chromosomal aberration assay or in vitro micronucleus assay or in vitro mouse lymphoma TK gene mutation assay  ASSAYS in vitro Bacterial reverse mutation assay (Ames assay) OECD Test No. 471 The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. Principle of the assay: The test employs essential amino acid deficient bacteria strains carrying different mutations in various genes of the histidine and tryptophan operon. These mutations act as hot spots for mutagens that cause DNA damage leading to mutations via different mechanisms. Gene mutations are detected when they cause restoration of the capability of the bacteria to synthesize the essential amino acid that are then able to grow in the absence of the amino acid required by the parent test strain. rfa mutations, a defective lipopolysaccharide layer that makes bacteria more permeable to larger molecules uvrB mutations, which eliminate excision repair of DNA damage  ASSAYS in vitro Bacterial reverse mutation assay (Ames assay) OECD Test No. 471 DESCRIPTION OF THE METHOD Suspensions of bacterial cells are exposed to the test substance (liquid or solid) in the presence and in the absence of an exogenous metabolic activation system. At least five different analysable concentrations of the test substance should be used. The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 mL/plate. There are two methods: the plate incorporation method and the preincubation method. For both techniques, after two or three days of incubation at 37°C, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.  ASSAYS in vitro Bacterial reverse mutation assay (Ames assay) OECD Test No. 471 Is compound X genotoxic?  ASSAYS in vitro Bacterial reverse mutation assay (Ames assay) OECD Test No. 471 CONSIDERATIONS The bacterial reverse mutation test utilises prokaryotic cells, which differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. In vitro metabolic activation systems cannot mimic entirely the mammalian in vivo conditions. There are examples of mutagenic agents which are not detected by this test. On the other hand, factors which enhance the sensitivity of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and those which are thought (or known) to interfere specifically with the mammalian cell replication system (e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test because they act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells  ASSAYS in vitro In vitro mammalian chromosomal aberration test OECD Test No. 473 The purpose of this test is to identify agents that cause structural chromosome aberrations in cultured mammalian somatic cells. Structural aberrations may be of two types: chromosome or chromatid. Principle of the assay: Cell cultures of human or other mammalian origin are exposed to the test chemical both with and without an exogenous source of metabolic activation unless cells with an adequate metabolizing capability are used. At an appropriate predetermined intervals after the start of exposure of cell cultures to the test chemical, they are treated with a metaphase-arresting substance (e.g. Colcemid or colchicine), harvested, stained and metaphase cells are analysed microscopically for the presence of chromatid-type and chromosome-type aberrations.  ASSAYS in vitro In vitro mammalian chromosomal aberration test OECD Test No. 473 DESCRIPTION OF THE METHOD A variety of cell lines (e.g. Chinese Hamster Ovary (CHO), Chinese Hamster lung V79, Chinese Hamster Lung (CHL)/IU, TK6) or primary cell cultures, including human or other mammalian peripheral blood lymphocytes, can be used. Exogenous metabolising systems should be used when employing cells which have inadequate endogenous metabolic capacity. At least three test concentrations should be evaluated. If no precipitate or limiting cytotoxicity is observed, the highest te.st concentration should correspond to 10 mM, 2 mg/mL or 2 µl/mL, whichever is the lowest. Cells should be exposed to the test chemical with and without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1.5 normal cell cycle lengths after the beginning of treatment. Afterwards the cells are treated with colcemid or colchicine usually for one to three hours prior to harvesting. Each cell culture is harvested and processed separately for the preparation of chromosomes. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining and then the inspection under the microscope follows.  ASSAYS in vitro In vitro mammalian chromosomal aberration test OECD Test No. 473 CONSIDERATIONS Tests conducted in vitro generally require the use of an exogenous source of metabolic activation unless the cells are metabolically competent with respect to the test substances. The exogenous metabolic activation system does not entirely mimic in vivo conditions. Care should be taken to avoid conditions that could lead to artifactual positive results, i.e. chromosome damage not caused by direct interaction between the test chemicals and chromosomes; such conditions include changes in pH or osmolality, interaction with the medium components or excessive levels of cytotoxicity. This test is used to detect chromosomal aberrations that may result from clastogenic events. The analysis of chromosomal aberration induction should be done using cells in metaphase. It is thus essential that cells should reach mitosis both in treated and in untreated cultures.  ASSAYS in vitro In vitro micronucleus test OECD Test No. 487 The in vitro micronucleus test is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells. Micronuclei are formed when either a chromosome fragment or an intact chromosome is unable to migrate to a mitotic pole during the anaphase stage of cell division and is left out of the daughter nuclei. The assay detects the activity of clastogenic and aneugenic test substances in cells that have undergone cell division during or after exposure to the test substance. Principle of the assay: Cell cultures are exposed to the test substances both with and without an exogenous source of metabolic activation unless primary cells with metabolizing capability are used. After exposure to the test substance, cell cultures are grown for a period sufficient to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells and to trigger the aneuploidy sensitive cell stage (G2/M). Harvested and stained interphase cells are then analysed microscopically for the presence of micronuclei.  ASSAYS in vitro In vitro micronucleus test OECD Test No. 487 DESCRIPTION OF THE METHOD Cultured cells from human peripheral blood lymphocytes or from Syrian Hamster Embryo (SHE) may be used. Exogenous metabolising systems are required when using cell cultures with inadequate endogenous metabolic capacity. If no cytotoxicity/cytostasis is observed when cells are treated with compound of interes, the highest concentration should correspond to 0.01 M, 5 mg/ml or 5 µl/ml, whichever is the lowest. To block cytokinesis Cytochalasin B is used to inhibit actin assembly and cytokinesis and thus prevents separation of daughter cells after mitosis and leads to binucleated cells. The use of a cytokinesis blocker is mandatory when human lymphocytes are used because cell cycle lengths will be variable within and between cultures. The appropriate concentration of cytochalasin B is usually between 3 and 6 µg/ml and should be added after the test substance is removed. Data indicate that most aneugens and clastogens will be detected by a short term treatment (3 - 6 hours). Cells are sampled at a time equivalent to about 2 times the normal cell cycle lengths after the beginning of treatment. Afterwards, cell culture is harvested, stained and microscopically analysed.  ASSAYS in vitro In vitro micronucleus test OECD Test No. 487 CONSIDERATIONS Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot entirely mimic in vivo conditions. Care should also be taken to avoid conditions that would lead to artefactual positive results which do not reflect intrinsic mutagenicity and may arise from e.g. changes in pH, osmolality or high levels of cytotoxicity. In order to analyse the induction of micronuclei it is essential that nuclear division has occurred in both treated and untreated cultures.  ASSAYS in vitro In vitro mammalian cell gene mutation assays based on the TK gene OECD Test No. 490 ▪ genetic events detected using the tk locus include both gene mutations and chromosomal events ▪ includes two distinct in vitro mammalian gene mutation assays requiring two specific tk heterozygous cells lines: → L5178Y (TK+/-) cells for the mouse lymphoma assay → TK6 (Tk+/-) cells for the human lymphoma assay. Principle of the assay: Cells deficient in thymidine kinase (TK-/- ) are resistant to the cytostatic effects of the pyrimidine analogue trifluorothymidine (TFT). TK proficient (TK+/-) cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain the TK enzyme, are not.  ASSAYS in vitro In vitro mammalian cell gene mutation assays based on the TK gene OECD Test No. 490 DESCRIPTION OF THE METHOD Cells in suspension or monolayer culture are exposed to, at least four concentrations of the test substance, both with and without metabolic activation, for a suitable period of time (usually 3 to 4 hours is adequate). They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection (2-4 days). Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.  ASSAYS in vitro In vitro mammalian cell gene mutation assays based on the TK gene OECD Test No. 490 CONSIDERATIONS Assays conducted using L5178Y TK+/- -3.7.2C or TK6 cells require the use of an exogenous source of metabolic activation. Care should be taken to avoid conditions that could lead to artifactual positive results (i.e. possible interaction with the test system) not caused by interaction between the test substance and the genetic material of the cell; such conditions include changes in pH or osmolality, interaction with the medium components or excessive levels of cytotoxicity. It should be noted that test chemicals that are thymidine analogues, or behave like thymidine analogues can increase the mutant frequency by selective growth of the spontaneous background mutants during cell treatment and require additional test methods for adequate evaluation  ASSAYS in vivo In vivo Mammalian Alkaline Comet Assay OECD Test No. 489 ▪ detects single or double strand breaks measured at the individual cell level; any tissue that can be dispersed to a single cell suspension can be used Principle of the assay: Animals are exposed to the test chemical by an appropriate route. At the selected sampling time(s), the tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in soft agar and exposed to strong alkali (e.g., pH≥13) to allow DNA unwinding and release of relaxed DNA loops and fragments. The nuclear DNA in the agar is then subjected to electrophoresis. Normal non-fragmented DNA molecules remain in the position where the nuclear DNA had been in the agar, while any fragmented DNA and relaxed DNA loops would migrate towards the anode. After electrophoresis, the DNA is visualized using an appropriate fluorescent stain. The extent of DNA that has migrated during electrophoresis and the migration distance reflects the amount and size of DNA fragments. The DNA content in the tail (% tail DNA or % tail intensity) has been recommended to assess DNA damage.  ASSAYS in vivo ICH S1A: Carcinogenicity studies should be performed for any pharmaceutical whose expected clinical use is continuous for at least 6 months. In addition, for pharmaceuticals used frequently in an intermittent manner in the treatment of chronic or recurrent conditions, carcinogenicity studies are generally needed.  ASSAYS in vivo Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays OECD Test No. 488 OECD adopted a test guideline on the use of transgenic rodent (TGR) assays for investigating the mutagenic potential of chemical agents. The TGRs used in these assays carry a transgene consisting of multiple copies of a bacterial gene which is incorporated into the animal genome. These transgenes have no effect on the animal but are easily recovered and tested for DNA mutations. These TGR assays have an advantage over bacterial and in vitro assays in that the exposure to the test agent occurs within a live animal with all of the various metabolic and DNA repair processes in place, thus more closely modeling actual human exposure. The possibility to investigate tissue specificity by examining DNA from various tissues in the same animal adds to the value of the assay. GENOTOXIC IMPURITIES ▪ a potential genotoxic impurity (PGI) has been defined as an “Impurity that shows a structural alert for genotoxicity but that has not been tested in an experimental test model. ▪ the main source of genotoxic impurities is the starting raw materials used for the manufacturing of drug substances, solvents, reagents, and catalysts. ▪ guidance is provided by the ICH Q3A- Impurities in New Drug Substance and Q3B (R2)- Impurities in New Drug Products Category/Stage Compounds Starting material Hydrazine, Nitroso, and acrylonitrile compounds Intermediate Benzaldehyde, Nitro compounds By-product Sulphonate esters, phosgene Reagent Formaldehyde, epoxides, esters of phosphate & sulphonates Solvent Benzene, 1,2-dichloroethane Catalyst Toxic heavy metals, metal phosphates Degradation product N-oxides, aldehydes, GENOTOXIC IMPURITIES Some examples of alerting functional groups that are known to be involved in reactions with DNA. GENOTOXIC IMPURITIES Different Classes of Potential or Real Mutagenic Impurities Based on Mutagenic and Carcinogenic Potential and Proposed Control Strategies. Impurity Class Commentary Control Strategy Control at or below compound’s specific 1 Known mutagenic carcinogens acceptable limit 2 Known mutagens with unknown carcinogenic potential Control at or below acceptable limits Control at or below acceptable limits Or Show alerting structures (un-related to drug substance) with no conduct bacterial mutagenicity assay; If 3 supporting mutagenicity data non-mutagenic = Class 5, If mutagenic = Class 2 Show alerting structures (related to drug substance which is Treat as a non-mutagenic impurity, i.e. 4 itself nonmutagenic) use default ICH Q3A/Q3B limits Treat as a non-mutagenic impurity, i.e. 5 Show no alerting structures use default ICH Q3A/Q3B limits  TAKE HOME MESSAGES ✓ recognition of various types of DNA damage, including chemical modifications, mutations, strand breaks, and crosslinks. ✓ appreciation of endogenous (internal) and exogenous (external) factors contributing to DNA damage ✓ understanding the link between DNA damage and the initiation of cancer diesease, which are hereogenous and complex ✓ knowledge on different classes of carcinogens ✓ insight into in vitro and in vivo testing methods, including genotoxicity assays to assess DNA damage, mutagenicity, and carcinogenicity and understanding the importance of these assays in regulatory assessments and safety evaluations for pharmaceuticals, chemicals, and other substances TAKE HOME MESSAGES