## 4.2.7. Chemical carcinogenesis and genotoxicity

Author: Timo Hamers

Reviewer: Frederik-Jan van Schooten

Learning objectives

You should be able to

- describe the three different phases in cancer development and understands how compounds can stimulate the corresponding processes in these phases
- explaion the difference between base pair substitutions and frameshift mutations both at DNA and the protein level
- describe the principle of bioactivation, which distinguishes indirect from direct mutagenic substances
- explain the difference between mutagenic and non-mutagenic carcinogens

Key words: Bioactivation; Mutation; Tumour promotion; Tumour progression; Ames test

Chemical carcinogenesis

Cancer is a collective name for multiple diseases sharing a common phenomenon that cell division is out of the control by growth-regulating processes. The consequent, autonomic growing cells are usually concentrated in a neoplasm (often referred to a tumour) but can also be diffusely dispersed, for instance in case of leukaemia or a mesothelioma. Benign tumours refer to neoplasms that are encapsulated and do not distribute through the body, whereas malign tumours cause metastasis , i.e. spreading of carcinogenic cells through the body causing new neoplasms at distant. The term benign sounds more friendly than it actually is: benign tumours can be very damaging to organs which are limited in available space (e.g. the brain in the skull) or to organs that can be obstructed by the tumour (e.g. the gut system).

The process of developing cancer (carcinogenesis) is traditionally divided in three phases, i.e.

1. the initiation phase, in the genetic DNA of a cell is permanently changed, resulting in daughter cells that genetically differ from their parent cells;
2. the promotion phase, in which the cell loses its differentiation and gains new characteristics causing increased proliferation;
3. the progression phase, in which the tumour invades surrounding tissues and causes metastasis.

Chemical carcinogenesis means that a chemical substance is capable of stimulating one or more of these phases. Carcinogenic compounds are often named after the phase that they affect, i.e. initiators (also called mutagens), tumour promotors, and tumour progressors. It is important to realize that many substances and processes naturally occurring in the body can also stimulate the different phases, i.e. inflammation and exposure to sun light may cause mutations, some endogenous hormones can act as very active promotors in hormone-sensitive cancers, and spontaneous mutations may stimulate the tumour progression phase.

Point mutations

Gene mutations (aka point mutations) are permanent changes in the order of the nucleotide base-pairs in the DNA. Based on what happens at the DNA level, point mutations can be divided in three types, i.e. a replacement of an original base-pair by another base-pair (base-pair substitution), the insertion of an extra base-pair or the deletion of an original base-pair (Figure 1). In a coding part of DNA, three adjacent nucleotides on a DNA strand (i.e. a triplet) form a codon that encodes for an amino acid in the ultimate protein. Because insertions and deletions cause a shift in these triplet reading frames with one nucleotide to the left or to the right, respectively, these point mutations are also called frame-shift mutations.

Based on what happens at the protein level for which a gene encodes, point mutations can also be divided into three types. A missense mutation means that the mutated gene encodes for a different protein than the wildtype gene, a nonsense mutation means that the mutation introduces a STOP codon that interrupts gene transcription resulting in a truncated protein, and a silent mutation means that the mutated gene still encodes for exactly the same protein, despite the fact that the genetic code has been changed. Silent mutations are always base-pair substitutions, because the triplet structure of the DNA has not been damaged.

Figure 1: Examples of missense, nonsense en silent mutations at the polypeptide level, based on base-pair substitutions en frame-shift mutations at the genomic DNA level.

A very illustrative example of the difference between a base-pair substitution and a frameshift mutation at the level of protein expression is the following “wildtype” sentence, consisting of only three letter words representing the triplets in the genomic DNA: The fat cat ate the hot dog. Imagine that the letter t in cat is replaced by an r due to a base-pair substitution. The sentence then reads: The fat car ate the hot dog. This sentence clearly has another meaning, i.e. it contains missense information. Imagine now that the letter a in fat is replaced by an e due to a base-pair substitution. The sentence then reads: The fet cat ate the hot dog. This sentence clearly contains a spelling error (i.e. a mutation), but it’s meaning has not changed, i.e. it contains a silent mutation. Imagine now that an additional letter m causes a frameshift in the word fat, due to an insertion. The sentence then reads: The fma tca tat eth eho tdo. This sentence clearly has another meaning, i.e. it contains missense information. Similarly, leaving out the letter a in fat also causes a frameshift mutation, due to a deletion. The sentence then reads: The ftc ata tet heh otd og. Again, this sentence clearly has another meaning, i.e. it contains missense information. This example suggests that the consequences are more dramatic for a frameshift mutation than for a base-pair substitution. Please keep in mind that the replacement of a cat by a car may also have huge consequences in daily life!

Mutagenic compounds

Base-pair substitutions are often caused by electrophilic substances that want to take up an electron from especially the nucleophilic guanine base that wants to donate an electron to form an electron pair. The consequent guanine addition product (adduct) forms a base-pair with thymine causing a base-pair substitution from G-C to A-T. Alternatively, the guanine adduct may split from the phosphate-sugar backbone of the DNA, leaving an “empty” nucleotide spot in the triplet that can be taken by any nucleotide during DNA replication. Alternatively, base-pair substitutions may be caused by reactive oxygen species (ROS), which are radical compounds that also take up an electron from guanine and form guanine oxidation products (for instance hydroxyl adducts). It should be realized that a DNA adduct can only cause an error in the order of nucleotides (i.e. a mutation) if it is present during DNA replication. Before a cell goes into the DNA synthesis phase of the cell cycle, however, the DNA is thoroughly checked, and possible errors are repaired by DNA repair systems.

Exposure to direct mutagenic electrophilic agents rarely occurs because these substances are so reactive that they immediately bind to proteins and DNA in our food and environment. Therefore, DNA damage by such substances in most cases originates from indirect mutagenic compounds, which are activated into DNA-binding agents during Phase I of the biotransformation. This process of bioactivation is a side-effect of the biotransformation, which is actually aiming at rapid detoxification and elimination of toxic compounds.

Frame-shift mutations are often caused by intercalating agents. Unlike electrophilic agents and ROS, intercalating agents do not form covalent bonds with the DNA bases. Instead, due to their planar structure intercalating agents fit exactly between two adjacent nucleotides in the DNA helix. As a consequence, they hinder DNA replication, causing the insertion of an extra nucleotide or the deletion of an original nucleotide in the replicate DNA strain.

Ames test for mutagenicity Mutagenicity of a compound can be tested in the Ames test, named after Bruce Ames who developed the assay in the early 1970s. The assay makes use of a Salmonella bacteria strain that contains a mutation in a gene encoding for an enzyme involved in the synthesis of the amino acid histidine. Consequently, the bacteria can no longer produce histidine (become “his‑”) and become auxotrophic, i.e. they depend on their culture medium for histidine. In the assay, the bacteria are exposed to the test compound in a medium that does not contain histidine. If the test compound is not mutagenic, the bacteria cannot grow and will die. If the test compound is mutagenic, it may cause a back-mutation (reversion) of the original mutation in a few bacteria, restoring the autotrophic capacity of the bacteria (i.e. their capacity to produce their own histidine). Growth of mutated bacteria on the histidine depleted medium can be followed by counting colonies (on an agar plate) or by measuring metabolic activity (in a fluctuation assay). Direct mutagenic compounds can be tested in the Ames test without extra treatment. Indirect mutagenic compounds, however, have to be bio-activated before they exert their mutagenic action. For this purpose, a liver homogenate is added to the culture medium containing all enzymes and cofactors required for Phase-I biotransformation of the test compound. This liver homogenate with induced cytochrome P450 (cyp) activity is usually obtained from rats exposed to mixed-type of inducers (i.e. cyp1a, cyp2b, cyp3a), such as the PCB-mixture Aroclor 1254.

Compounds involved in tumour promotion and tumour progression

As stated above, non-mutagenic carcinogens are involved in stimulating the tumour promotion. Tumour promoting substances stimulate cell proliferation and inhibit cell differentiation and apoptosis. Unlike mutagenic compounds, tumour promoting compounds do not interfere directly with DNA and their effect is reversible. Many endogenous substances (e.g. hormones) may act as tumour promoting agents.

The first illustration that chemicals may induce cancer comes from the case of the chimney sweepers in London around 1775. The surgeon Percival Pott (1714-1788) noticed that many adolescent male patients who had developed scrotal cancer had worked during their childhood as a chimney sweeper. Pott made a direct link between exposure to soot during childhood and development of cancer at later age. Based on this discovery, taking a shower after work became mandatory for children working as chimney sweepers, and the observed scrotum cancer incidence decreased. As such, Percival Pott was the first person (i) to link cancer development to chemical substances, (ii) to link early exposure to later cancer development, and (iii) to obtain better occupational health by decreased exposure through better hygiene. In retrospective, we now know that the mutagens involved were polycyclic aromatic hydrocarbons (PAHs) that were bio-activated into highly reactive diol-epoxide metabolites. The delay in cancer development after the early childhood exposure can be attributed to the absence of a tumour promotor. Only after the chimney sweepers had gone through puberty they had sufficient testosterone levels, which stimulates scrotum tissue growth and in this case acted as an endogenous tumour promoting agent.

Tumour progression is the result of aberrant transcriptional activity from either genetic or epigenetic alterations. Genetic alterations can be caused by substances that damage the DNA (called genotoxic substances) and thereby introduce strand breaks and incorrect chromosomal division after mitosis. This results in the typical instable chromosomal characteristics of a malign tumour cell, i.e. a karyotype consisting of reduced and increased numbers of chromosomes (called aneuploidy and polyploidy, respectively) and damaged chromosomal structures (abberations). Chemical substances causing aneuploidy are called aneugens and substances causing chromosomal abberations are called clastogens. Genotoxic substances are also very often mutagenic compounds. Multiple mutations in so-called proto-oncogenes and tumour suppressor genes are necessary to transform a normal cell into a tumour cell. In a healthy cell, cell proliferation is under control by proto-oncogenes that stimulate cell proliferation and tumour suppressor genes that inhibit cell proliferation. In a cancer cell, the balance between proto-oncogenes and tumour suppressor genes is disturbed: proto-oncogenes act as oncogenes, meaning that they continuously stimulate cell proliferation, due to mutations and polyploidy, whereas tumour suppressor genes have become inactive due to mutations and aneuploidy.

Epigenetic alterations are changes in the DNA, but not in its order of nucleotides. Typical epigenetic changes include changes in DNA methylation, histone modifications, and microRNA expression. Compounds that change the epigenome may stimulate tumour progression for instance by stimulating expression of oncogenes and inhibiting expression of tumour suppressor genes. The role in tumour promotion and progression of substances that are capable to induce epigenetic changes is a field of ongoing study.