PRINCIPLES OF TOXICOLOGY - Biology East Borneo

12.2 GENETIC FUNDAMENTALS AND EVALUATION OF GENETIC CHANGE 247the number of bases is unchanged but the sequence is altered. Because the genetic code is “degenerate,”this may or may not result in an altered product after transcription and translation. A frameshiftmutation, however, results from insertion or deletion of one or more bases from the linear sequenceof the DNA. This causes the transcription process to be displaced by the corresponding number ofbases and virtually assures an altered genetic product. Proflavine, which has been used as a bacteriostaticagent, is an example of a compound that intercalates within the DNA molecule. It is a flat,planar molecule and inserts itself neatly between the bases. When it intercalates, it forces the DNAstrand out of its normal configuration, so that when the replication enzymes or transcription enzymestry to read the bases, the bases are not spatially arranged the normal way, and the enzymes cannot readthe base sequence properly. The enzymes may skip over one or several bases, or may put an additionalbase into the DNA or RNA strand at random. Proflavine does not chemically bind with the bases inDNA. In contrast, many of the environmentally prevalent polynuclear aromatic hydrocarbons (PAHs)may intercalate into the DNA, leading to frameshift, and also may chemically react directly with it, anevent that can lead to basepair substitution. An example of this is benzo(a)pyrene (BaP), which is foundat low concentrations throughout the environment as a product of combustion of fossil fuels, in grilledsteaks, tobacco smoke, and many other places. BaP by itself is seldom considered to be mutagenic.However, after metabolism, many highly reactive epoxide intermediate metabolites are formed, oneof which (BPDE I) is highly mutagenic. BPDE I combines with guanine to form what is called a DNAadduct. These adducts have been found in extremely small quantities by highly specialized andsensitive techniques such as enzyme-linked immunosorbent assay (ELISA) and fluorescence. Ascheme of activation and adduct formation for BaP is given in Figure 12.5.Basepair changes, described earlier, are of two kinds: transitions or transversions. In transitions,one base is replaced by the base of the same chemical class. That is, a purine is replaced by the otherpurine (e.g., adenine is replaced by guanine); in the case of pyrimidine bases, cytosine would bereplaced with thymine or vice versa. An example of a chemical that causes transitions is nitrous acid(see Figure 12.6). Nitrous acid is formed from organic precursors such as nitrosamines, nitrite, andnitrate salts. It reacts with amino (NH 2) groups in nucleotides and converts them to keto (C?O) groups.In transversions, a base pair is replaced in the DNA strand by a base of the other type: a purine isreplaced by a pyrimidine or vice versa.Another group of chemicals that can cause mutations are alkylating agents. Some well-knownalkylating agents are the mustard gases, originally developed for chemical warfare. Chemicals in thisgroup add short carbon–hydrogen chains at specific locations on bases. The experimental agent ethylmethanesulfonate (EMS) can alkylate guanine to form 7-ethylguanine (see Figure 12.7), which cancause the bond between the base and deoxyribose in the backbone of the DNA strand to becomeunstable and break. This leads to a gap in the DNA strand which, if unrepaired at the time of DNAreplication, is filled with any of the four available bases.Not all point mutations are caused by radiation or chemicals; some may occur because of the natureof the bases themselves. The bases have their preferred arrangement of hydrogen atoms, but on rareoccasions undergo rearrangements of the hydrogen atoms, called tautomeric shifts. The nitrogen atomsattached to the purine and pyrimidine rings are usually in the amino (NH 2) form and only rarely assumethe imino (NH) form. Similarly, the oxygen atoms attached to the carbon atoms of guanine and thymineare normally arranged in the keto (C?O) form, but rarely rearrange to the enol (COH) form.The changes in configuration lead to different hydrogen bonding patterns, and, if a base is in thealternate form during replication, a wrong base can be put into the new growing strand leading to amutation. A group of chemicals, base analogs, that resemble the normal bases of DNA may lead tomutations by being incorporated into DNA inadvertently during repair or replication. These chemicalsgo through tautomeric shifts more often and result in inappropriate base pairing during replication sothat changes in the base sequence occur. An example of a base analogue is 5-bromouracil, which canreplace thymine.Gene mutation tests measure those alterations of genetic material limited to the gene unit, that aretransmissible to progeny unless repaired. Brusick (1980) refers to gene mutations as “microlesions”because the actual genetic lesion is not microscopically visible. Microlesions are classified as either