Mechanism of action of antitumor drug etoposide: a review.

Metabolism studies of the antitumor drug etoposide show the formation of metabolites in the lactone ring, which are probably not important for the drug's mechanism of action, and oxidative transformations in the dimethoxyphenol ring (E ring), which lead to products that can cause DNA damage and may play a role in the drug's mechanism of action. The cytotoxicity of etoposide is caused by the induction of DNA damage. The occurrence of the DNA lesions can be explained by the capacity of the drug to interfere with the scission-reunion reaction of mammalian topoisomerase II by stabilizing a cleavable complex.

Cytochrome P-450-mediated O-demethylation: a route in the metabolic activation of etoposide (VP-16-213).

The antitumor agent VP-16-213 is oxidatively O-demethylated by rat liver microsomes and purified rat liver microsomal cytochrome P-450. 3-Methylcholanthrene can quantitatively induce O-demethylation of VP-16-213. The Km and Vmax values for O-demethylation by noninduced, phenobarbital-, and 3-methylcholanthrene-induced rat liver microsomes were found to be 130, 600, and 160 microM and 8.5, 11.8, and 15.6 nmol H2CO/min X mg protein, respectively. Mass spectrometric comparison of the product of O-demethylation of VP-16-213 with the synthetic metabolite resulted in identification of the orthodihydroxy derivative. In studies on the biological activity of the orthodihydroxy derivative, it was found to inactivate single- and double-stranded phiX174 DNA, to bind to calf thymus DNA and to be highly toxic against chinese hamster ovary cells.

Effects of adduct formation on the biological activity of single- and double-stranded øX174 DNA, modified by N-acetoxy-N-acetyl-2-aminofluorene.

In order to establish a good quantitative relationship between the number of acetylaminofluorene adducts and the extent of inactivation of DNA, single-stranded (ss) øX174 DNA and øX174 RF DNA were modified to various extents with 3H-labelled N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) and subsequently transfected to Escherichia coli spheroplasts having different repair capabilities. Exponential survival curves were obtained. In the case of ssDNA about one adduct per molecule appears to be lethal. On the other hand only 1 out of 10.2 adducts is found to inactivate RF DNA if tested on wild-type E. coli. However, when assayed on strains deficient in excision repair 1 out of 2.3 adducts leads to inactivation of RF DNA. RecA-dependent postreplication repair only has little influence on these figures. Product analysis of the modified DNAs shows that in RF DNA at least 76% of the interaction products is N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (dGuo-C8-AAF) and at least 6% and at most 12% is 3-(deoxyguanosin-N2-yl)-N-acetyl-2-aminofluorene (dGuo-N2-AAF). In ssDNA only dGuo-C8-AAF is formed. No apurinic sites could be detected in the modified DNAs. From these results it can be concluded that in RF DNA most of the dGuo-C8-AAF is removed by excision repair. The remaining damage, consisting probably both of dGuo-N2-AAF and unexcised dGuo-C8-AAF, inactivates RF DNA. Inactivation can be explained by a model which shows that only damage in the minus strand of RF DNA inhibits replication and/or transcription.

Structure and transcription specificity of yeast RNA polymerase A.

DNA-dependent RNA polymerase A (Nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was isolated from whole yeast cells and purified to a nearly homogeneous state. The subunit structure as well as the transcription specificity of the purified enzyme were investigated. Polyacrylamide gel electrophoresis under denaturating conditions revealed that yeast polymerase A is made up of two large subunits having mol. wts of 190 000 and 135 000, and five smaller subunits with mol. wts of 54 000, 44 000, 35 000, 25 000 and 16 000, respectively. The molar ratios of all these polypeptides were found to be about unity. The transcription specificity of yeast polymerase A was tested using homologous nuclear DNA as a template. The in vitro synthesized RNA was characterized by determining its degree of self-complementarity and its ability to compete with purified ribosomal RNA in hybridization experiments. It was found that yeast polymerase A is capable of a highly selective transcription in vitro of the rRNA cistrons, provided DNA of high integrity is used as a template.

Modulation by D,L-buthionine-S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines.

Treatment with 25 mumol/l D,L-buthionine-S,R-sulphoximine (BSO) for at least 24 h depleted glutathione (GSH) in human non-small cell lung (SW-1573), ovarian (A2780) and breast carcinoma (MCF-7) cell lines to about 20% of control, and was accompanied by a 2-fold potentiation of the cytotoxicity of etoposide, doxorubicin and cisplatin. Cellular etoposide, but not doxorubicin or cisplatin, concentrations were increased 2-fold due to decreased efflux. This occurred independently of the presence of BSO during 1 h of etoposide exposure, but required prolonged exposure to BSO (at least 24 h). Energy depletion as well as cotreatment, but not pretreatment, of the cells with daunomycin, doxorubicin, vinblastine or vincristine increased cellular etoposide accumulation. Treatment of control cells with verapamil caused similar changes in etoposide cytotoxicity and cellular pharmacokinetics as GSH depletion, but did not further increase etoposide cytotoxicity and accumulation in GSH-depleted cells. Etoposide efflux may have been inhibited, not because of (competitive) inhibition by BSO or disturbance of the energy required for this process, but probably because of plasma membrane alterations.

Interaction of RSU 1069 and 1137 with DNA in vitro. Biological implications and mechanistic aspects.

We have examined the capacity of the nitroimidazole aziridine antitumour drug RSU 1069 to react with DNA in vitro in order to get a better understanding of its mechanism of action. Moreover, we have utilized biologically active phi X174 DNA to investigate the biological relevance of the chemical DNA modification induced by the drug. Incubation of RSU 1069 in the presence of single-stranded phi X174 DNA resulted in extensive inactivation of the DNA, which is dependent on the concentration of drug and temperature. Only about 2% of the inactivating damage can be attributed to strand breakage. The main damage most probably consists of base damage, of which a part is non-lethal and alkali-labile which in turn can be converted into lethal lesion and subsequently into a break applying a post-incubation alkali treatment. Furthermore, from the dependence of the inactivation and also the formation of breaks on pH and ionic strength, it is concluded that the reaction most probably takes place between a protonated RSU 1069 and a negative DNA coil and that the damage pattern reflects the difference in reactivity of RSU 1069 with the phosphate groups and the bases in DNA. Comparison between RSU 1069 and its ring-open hydrolysis product RSU 1137 revealed that (lethal) damage induced in the DNA must be ascribed to the alkylating properties of the aziridine moiety.

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213).

In this report, the types of DNA damage introduced by the ortho-quinone and the semiquinone free radical of 4'-demethylepipodophyllotoxin-9-(4-6-O-ethylidene-beta-D- glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) phi X174 DNA have been examined in vitro. The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF phi X174 DNA, respectively. The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation. The radical introduced, in addition, chemical DNA adducts. Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss phi X174 DNA, while more than 40 were required for the inactivation of RF DNA. The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts [van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retèl J and Pinedo HM, Biochem Pharmacol 37: 3579-3589, 1988]. When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced. In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss phi X174 DNA, resulted in a partial restoration of the biological activity. It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity.

Reactions of glutathione with the catechol, the ortho-quinone and the semi-quinone free radical of etoposide. Consequences for DNA inactivation.

Etoposide [4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta- D-glucopyranoside)] can be metabolized to DNA-inactivating catechol, ortho-quinone and semi-quinone free radical derivatives which may contribute to its cytotoxicity. In this paper, we examined in vitro whether glutathione (GSH), which is known to react easily with quinoid compounds, could interact with the active etoposide intermediates and in this way influence the cytotoxicity of the parent compound. To this end, reactions of GSH with the etoposide intermediates were studied, using HPLC and ESR measurements, together with the effects of GSH on the biological inactivation of single-stranded (ss) and double-stranded (RF) phi X174 DNA by these compounds. From the results it could be determined that: (a) GSH does not react with the catechol and, as a consequence, has no effect on the reaction of this intermediate of etoposide with ss and RF phi X174 DNA; (b) GSH reacts with the ortho-quinone most likely by formation of a conjugate and by two-electron reduction to the catechol, resulting in a partial protection of ss and RF phi X174 DNA against inactivation by this species; and (c) GSH protects ss phi X174 DNA against inactivation by the semi-quinone free radical of etoposide probably by conjugation with this species.

Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded phi X174 DNA.

We have studied the effects of the recently reported two new metabolites of the antitumor agent VP-16-213, the ortho-dihydroxy derivative or catechol and the ortho-quinone, on the biological activity of single-stranded and double-stranded phi X174 DNA, the binding of the metabolites to calf thymus DNA and the conversion of the catechol into the ortho-quinone. Evidence was obtained for the oxidation of the catechol into the ortho-quinone and for the fact that the ortho-quinone is the metabolite of VP-16-213 responsible for its binding to rat liver microsomal proteins. The catechol and ortho-quinone of VP-16-213 were found to bind 7-9 times more strongly to calf thymus DNA than VP-16-213 itself. In contrast to the parent compound VP-16-213, the catechol as well as the ortho-quinone inactivated both single-stranded (ss) and double-stranded (RF) biologically active phi X174 DNA. The mean T37-values for inactivation of ss and RF phi X174 DNA by 2.2 x 10(-4)M catechol at 37 degrees and pH 7.4 were 96 and 640 min, respectively. Reduction of the ortho-quinone by NADPH cytochrome P-450 reductase resulted in formation of the catechol. The system ortho-quinone/NADPH cytochrome P-450 reductase inactivated ss phi X174 DNA with a mean T37-value of 454 min, and this inactivation was inhibited by DMSO. The mean T37-value for inactivation of ss phi X174 DNA by 1.8 x 10(-4) M ortho-quinone at 37 degrees and pH 4.0 was 24 min. The chemical stability of the ortho-quinone and the extent of inactivation of ss phi X174 DNA by the ortho-quinone were both pH-dependent: at higher pH the ortho-quinone was less stable and gave less inactivation of DNA. The aqueous decomposition product(s) of the ortho-quinone formed at pH 7.4 inactivated ss phi X174 DNA with a mean T37-value of 175 min. The rate of inactivation of RF phi X174 DNA by the ortho-quinone at pH 4.0 was twice as low as the rate of inactivation of ss phi X174 DNA: T37 = 49 min. When using excision repair deficient E. coli mutants (uvrA- or uvrC-), a higher inactivation of RF phi X174 DNA was found: T37 = 29 min for uvrA- E. coli, indicating that a part of the DNA damage introduced by the incubation with ortho-quinone is removed by excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)

The ambivalent role of glutathione in the protection of DNA against singlet oxygen.

Glutathione (GSH) was examined with respect to its ability to protect DNA against 1O2 damage. We have found that GSH protected, at least partly, the DNA against inactivation by 1O2. Up to 10 mM the protection increased as a function of GSH concentration. Above 10 mM the protection remained constant and less than expected on the basis of scavenging/quenching of 1O2, in contrast to the protection offered by sodium-azide. Especially at the higher concentrations of GSH the protection against the biological inactivation is accompanied by an increase in single-strand breaks and also probably lethal base damage. However, all together the data suggest that at least in the physiologically important range (0.1-10 mM) GSH is able to protect efficiently against 1O2-induced inactivating DNA damage.

Excited state properties of the N-(deoxyguanosin-8-yl)-2-aminofluorene adducts.

The spectroscopic characteristics of adducts derived from the covalent binding of the carcinogen 2-aminofluorene to the C8 position of deoxyguanosine [N-(deoxyguanosin-8-yl)-2-amino-fluorene, dGuo-C8-AF], and from an adduct of similar structure formed with the synthetic polynucleotide poly(dG-dC).poly(dG-dC), were investigated. At 77 K both adducts are characterized by well-defined and rather narrow fluorescence emission spectra with maxima at 370 and 390 nm characteristic of the aromatic, monomolecular 2-aminofluorene (AF) residue. In contrast, at room temperature, the fluorescence is characterized by a broad, structureless emission band with a maximum at 460 nm in aqueous mixtures, shifting to 415 nm in solvents of lower polarity (100% propanol); the maxima are located at intermediate wavelengths in solutions of different propanol/water compositions, and this emission is attributed to an excited state complex (exciplex). The fluorescence quantum yield decreases when either the solvent polarity or the temperature are increased, varying from 5.4% (100% propanol) to 0.04-0.05% (100% H2O). The fluorescence decay profiles of dGuo-C8-AF adducts (measured at the National Synchrotron Light Source facility at the Brookhaven National Laboratory) can be roughly, but not exactly, modeled in terms of two exponential decay components in the range of about 0.3-1.0 ns with the propanol concentration greater than 60%; at lower propanol concentrations, a single short lifetime is observed and in 100% water solutions its value is 0.08 ns. The shorter lifetime, favored in solvent mixtures of higher polarities, is attributed to an exciplex with significant charge-transfer character.(ABSTRACT TRUNCATED AT 250 WORDS)

A circular dichroism study on the conformation of d(CGT) modified with N-acetyl-2-aminofluorene or 2-aminofluorene.

The trinucleotide d(CGT) was modified by covalent binding of the carcinogen N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) at the C8 position of the guanine base. The conformations of d(CGT)-AAF and -AF were studied by comparing the absorption and circular dichroism properties with those of dCMP + dGMP-AAF or -AF + dTMP in a molar ratio of 1:1:1 and AAF- and AF-containing dGMP. For both AAF- and AF-d(CGT) complexes the results show significant stacking interactions between the fluorene residue and the base(s) and are discussed in terms of the conformation of d(CGT)-AAF and -AF. In d(CGT)-AF we observe a clear interaction between AF and thymine, whereas the C-G stack is still intact. In the case of d(CGT)-AAF the C-G stack is weakened and the glycosidic rotation angle of dGuo-C8-AAF is most probably syn. The specific fluorene-base interactions persist at elevated temperatures. The carcinogen-base interactions are stronger in the AAF-carrying d(CGT) than in the case of the deacetylated complex. This is consistent with the higher mobility of the AF-adduct and its conformationally heterogeneous appearance in DNA.

Mutations induced by gamma-irradiation of M13 bacteriophages containing single-stranded DNA.

Oxygenated suspensions of M13 bacteriophages, containing single-stranded M13mp10 DNA, were gamma-irradiated followed by infection of E. coli cells. Mutants in the mutational target sequence, which consists of the lac promoter /operator region, the lacZ alpha gene, and a 144 bp inframe insert in the lacZ alpha gene, were selected and characterized. Except for three one-base deletions, all of the 51 mutations characterized were base substitutions. All base substitutions appeared to involve guanines and cytosines and none affect adenines and thymines. Since most of the known repair systems do not act on single-stranded DNA, the conclusion can be drawn that radiation induces under these conditions only mutagenic damages on guanine and cytosine. Although all possible G- and C-transversions and transitions were found, there is a strong preference for G-->C and G-->T transversions (21 and 25% of all base substitutions, respectively) and C-->T transitions (48% of all base substitutions). These results indicate, that the G/C-->C/G and G/C-->T/A transversions, found after irradiation of double-stranded M13 DNA, are mainly due to radiation guanine products, whereas cytosine damage is mainly responsible for G/C-->A/T transitions.

Mutations induced by 60Co gamma-irradiation in double-stranded M13 bacteriophage DNA in nitrous-oxide saturated solutions are characterized by a high specificity.

Irradiation of double-stranded M13 mp10 DNA in a diluted aqueous solution under N2O leads to a very specific mutation spectrum. Fifteen of 28 mutations induced in a 144 base pair (bp) target are C/G to G/C transversions, the other five bp substitutions are C/G to A/T transversions. Six mutations were single bp deletions, one is a large deletion of 180 bp and one is a 10 bp duplication which is probably from spontaneous origin. The mutations are not randomly distributed throughout the 144 bp mutation target but concentrated around two sites. The differences and similarities with the radiation-induced mutation spectrum previously obtained under oxygen are discussed.

Inactivation and repair of double-stranded DNA damaged by the aziridinyl nitroimidazole RSU 1069.

Incubation of RSU 1069 in the presence of biologically active double-stranded phi X174 DNA resulted in, depending on pH, ionic strength and concentration of drug, inactivation of the DNA. A variety of lesions are induced including a high number of single-strand breaks and alkali-labile lesions, which are at most partly lethal. The main inactivating damage consists probably of base damage, induced by alkylation. A considerable part of the damage induced by RSU 1069 can be repaired by the various repair enzymes of the bacterial host of the phi X174 DNA. Finally the damage (pattern) depends considerably on the ionic composition of the reaction solution, which can be explained by an equilibrium model presented in this paper.

Contrasting effects of SH-compounds on oxidative DNA damage: repair and increase of damage.

The non-radical singlet oxygen (1O2) and the OH radical (.OH) are the major damaging oxidative species that can be generated inside cells during normal aerobic metabolism and by processes such as photosensitization. Both reactive oxygen species fulfill essential prerequisites to be a genotoxic agent. Due to their continuous production they represent an ever-present threat to all vital cellular molecules, especially DNA. As might be anticipated from the difference in character between these reactive species (non-radical versus radical) the pattern of DNA modifications caused by singlet oxygen is different from that produced by OH radicals. All cells possess an elaborate defense system against oxidative damage. This paper focuses mainly on the effect of thiols such as glutathione, which are thought to play a role as antioxidants. Under certain conditions thiols can repair chemically, probably by H-donation, some of the DNA damage caused by .OH; for instance breaks can be rather easily prevented in this way. This process will compete with fixation of damage by oxygen. However, there is ample evidence that H-atom donation does not always lead to 'correct' repair. Moreover under aerobic conditions thiyl peroxy radicals might increase DNA damage. Although the repair/fixation process could not be examined in the case of 1O2 yet, it could be demonstrated that reactive species can be formed out of the reaction of thiols with 1O2 capable of enhancing the number of DNA modifications such as 8-oxoguanine and single-strand breaks, probably arising from different pathways. Although it is quite clear that thiols are to some extent excellent antioxidants they possess unexpected properties which, depending on the conditions, can have genotoxic consequences.

Effect of the sulfhydryl compound cysteamine on gamma-radiation-induced mutations in double-stranded M13 DNA.

Sulfhydryl compounds can protect DNA against free-radical-induced DNA damages not only by scavenging of radicals, but also by chemical non-enzymatic repair or modification of such damages by hydrogen-donation. To investigate the influence of chemical repair and modification on mutations, induced by gamma-radiation-generated free radicals (.OH, .H), phosphate-buffered aqueous solutions of double-stranded (ds) M13 DNA were exposed to gamma-rays under N2 in the presence of 5 mM cysteamine. The exposed DNA was subsequently transfected to wild-type E. coli and mutations in the mutational target were characterized. This target in fact contains three different target sequences, i.e., the lac promoter/operator, the lacZ alpha gene and a 144 bp inframe insert. The mutation spectrum obtained was compared with those in the absence of cysteamine under N2 and N2O. In the latter case, the ratio of .OH and .H available for reacting with DNA is about the same as under N2 + cysteamine. The results show that chemical repair and/or modification by cysteamine of potentially lethal lesions takes place, leading to a much higher survival of ds M13 DNA in the presence of cysteamine than could be expected on basis of scavenging of .OH and .H alone. This higher survival appeared to be accompanied with a higher mutation induction. However, the N2 + cysteamine mutation spectrum shows a remarkable resemblance with the N2O-spectrum. This holds for the total mutation target, as well as each of the three targets, although the mutations obtained in each of the three targets under the same irradiation conditions are quite different. Thus, it can be concluded that cysteamine is mainly effective on radiation-induced potentially lethal DNA lesions, and not so much on (pre)mutagenic damages. Moreover, the type of mutation appeared to be strongly dependent on the mutational target sequence.

C/G to A/T transversions represent the main type of mutation induced by gamma-irradiation in double-stranded M13mp10 DNA in a nitrogen-saturated solution.

To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays. Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH). The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene. A very specific mutation spectrum was obtained with respect to the type of mutations. Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion. The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots. When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions.

The formation of one-G deletions as a consequence of single-oxygen-induced DNA damage.

Single-stranded M13mp10 DNA containing a 144-bp mutational target sequence in the lacZ alpha gene was treated with singlet oxygen (1O2) generated by thermodissociation of the endoperoxide of 3,3'-(1,4-naphthalene-1,4-diyl)dipropionate (NDPO2). After transfection to non-SOS-induced E. coli cells, 32 mutants preselected for a mutation in the 144-bp target were collected and analyzed by DNA sequencing. One-G deletions represented the predominant type of mutation accounting for 50% of the mutations analyzed. The remaining part appeared to consist of base substitutions, i.e. G-->T transversions (34%), C-->T transitions (12.5%) and one T-->C transition (3%). Sixty percent of the mutations were found in two major mutational hotspots. We conclude that the predominant one-G deletions are due to a guanine reaction product which might be specific for 1O2.

Gamma-radiation-induced mutation spectrum in the episomal lacI gene of Escherichia coli under oxic conditions.

In this study we have determined the mutation spectrum in the complete episomal lacI gene of Escherichia coli induced by gamma-radiation under oxic conditions. Mutants were generated by 60Co gamma-irradiation of an E. coli culture of stationary cells in LB medium, under continuous flushing with oxygen. Oligonucleotide probe analysis showed that 14% of the gamma-ray-induced mutations were located at the lacI gene hot spot at position 620-632, which is characterized by a triple repeat of the 5'-TGGC-3' sequence. Previously it was shown that about 70% of the spontaneous mutations were located at this site due to the loss or the addition of a TGGC sequence. The non-hot spot mutations were further characterized by automated sequence analysis. The results show that base pair (bp) substitutions were the main type of gamma-ray-induced mutations. Although all types of bp substitutions were observed, 74% of the bp substitutions involved C/G base pairs. C/G --> T/A and C/G --> A/T substitutions were predominant, both accounting for 35% of all bp substitutions, whereas A/T --> C/G substitutions were only seldomly observed (3%). A relatively large amount of -1 bp deletions (15% of all mutations) was detected in the gamma-ray-induced mutation spectrum, mainly affecting C/G base pairs, and 10% were deletions, ranging in size from 11 to 532 bp. It can be concluded that under oxic conditions gamma-radiation induces in E. coli mainly bp substitutions of all types but preferentially at C/G base pairs, and that the mutations tend to be randomly distributed within the lacI gene sequence.