Relationships between structure, toxicity and genetic effects in Salmonella typhimurium and Saccharomyces cerevisiae for substituted aniline mustards

A series of 4-substituted aniline mustards of widely varying reactivities have been evaluated for their mutagenic effects in Salmonella typhimurium strains of varying uvrB gene and plasmid status, and for their ability to cause mitotic crossing-over in Saccharomyces cerevisiae. The 4-methyl aniline mustard N,N-bis(2-chloroethyl)-4-methylaniline and its corresponding half-mustard N-(2-chloroethyl)-4-methylaniline showed widely different effects in the various bacterial strains, with the half-mustard being much less toxic than the full mustard in the uvrB- strain TA100. However, in the uvrB+ strain TA1978+, possessing an intact excision repair system, both compounds were equally toxic and the full mustard was the more mutagenic. Both compounds were equally effective in promoting mitotic crossing-over in yeast. For a series of 4-substituted full mustards, the toxicity in S. typhimurium strain TA100 correlated with substituent electronic parameters in the same way as does mammalian cell toxicity, supporting the view that the primary mode of toxicity is via DNA cross-linking, even for unreactive analogues. However, there were no obvious correlations between substituent physiochemical properties and mutagenic potential in bacteria, suggesting that mutagenic events are subject to a variety of influences other than the reactivity of the mustard group. In contrast, the most chemically reactive compounds were the most toxic and most recombinogenic in yeast.     

Mutagenic activities of azido analogues of amsacrine and other 9-anilinoacridines in Salmonella typhimurium and their enhancement by photoirradiation.

Analogues of amsacrine and other 9-anilinoacridines, bearing azide groups at various positions, were tested for their mutagenic activity in five strains of Salmonella typhimurium, both before and after photoirradiation. Azido substitution at the 2- or 3-position of the acridine or the 1'-position of the aniline led to compounds which were active frameshift mutagens, as detected in strain TA1537. Photoirradiation enhanced both the mutagenicity and the cytotoxicity of the azido compounds. Analogues bearing two azido groups at either the 2,6- or 3,6-positions were less strongly mutagenic in the dark, and light activation led to a toxic but only weakly mutagenic product. The effects of photoirradiation were decreased by aniline ring substitution, and were essentially eliminated by additional methyl substitution in the acridine ring. Comparison of events in TA1537 and TA98 suggested that photoirradiation of the 2- or 3-azido compounds gave a product which was capable of forming covalent bonds with DNA. The azide-containing analogues readily formed single strand DNA breaks on irradiation in the presence of DNA, but the efficiency of this reaction varied considerably.     

重叠延伸PCR对DNA片段进行定点双突变

探讨如何利用重叠延伸PCR对同一靶DNA片段中的两个不同位点实施联合突变。先用野生型DNA作模板,通过一轮重叠延伸PCR,获得突变一个预期位点的DNA片段,再用此突变DNA片段作模板,通过另一轮重叠延伸PCR获得两个预期位点均突变的DNA片段。重叠延伸PCR能对DNA片段进行双突变甚至多点突变,具有简便、快速、经济等特点,在阐明基因的调控机理、改造蛋白质结构等分子生物学领域中具有极大的应用价值。     

Mutagens formed from beta-carbolines with aromatic amines

Norharman, widely distributed in our environment such as cigarette smoke and cooked foods, is not mutagenic to Salmonella strains, but becomes mutagenic to Salmonella typhimurium TA98 and YG1024 with S9 mix in the presence of aromatic amines, including aniline and o-toluidine. Therefore, we have designated norharman as a "co-mutagen". Since, humans are simultaneously exposed to norharman and aromatic amines in daily life, it is important to clarify the mechanisms of its co-mutagenic action to further understanding of the potential genotoxic effects in humans. Regarding the mechanisms of this action of norharman with aniline, a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole[aminophenylnorharman (APNH)] is produced by their interaction, and converted to the hydroxyamino derivative which eventually forms the DNA adduct, dG-C8-APNH through possible ultimate reactive forms with esterification, and this induces mutations. Also other aminophenyl-beta-carboline compounds, such as 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole[amino-3'-methylphenylnorharman (3'-AMPNH)], 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole [amino-2'-methylphenylnorharman (2'-AMPNH)], 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole[aminophenylharman (APH)] and 9-(4'-amino-3'-methylphenyl)-1-methyl-9H-pyrido[3,4-b]indole[amino-3'-methylphenylharman (AMPH)], have been found on reaction of norharman or harman with aniline or toluidine isomers. These compounds showed mutagenic and clastogenic actions in bacterial and mammalian cells. Among them, APNH demonstrated the most potent activity, and it was most extensively studied. When APNH was administered as a single dose to F344 rats, severe testicular toxicity was observed after 6 days. Moreover, liver preneoplastic lesions (GST-P-positive foci) in the liver clearly developed in animals fed 10-50 ppm of APNH in the diet for 4 weeks. Since, APNH was detected in 24 h urine of rats upon simultaneous administration with norharman and aniline by gavage, it is likely to be also produced from norharman and aniline in the human body. From these findings, it is suggested that aminophenyl-beta-carboline derivatives may be classified as one of the novel types of endogenous mutagens and carcinogens.     

The implications of using mutagenic primers in combination with Taq polymerase having proofreading activity.

Polymerases with proofreading activity provide high fidelity PCR amplifications. In this study we examined the consequences of using a Taq polymerase with proofreading activity, such as Optimase Taq polymerase, in combination with 4 different mutagenic reverse primers for the amplification of a 345-bp FII PCR product. The amplifications were performed with Optimase Taq polymerase (Transgenomic), and Taq DNA polymerase-recombinant (Invitrogen), without proofreading activity. Mutation screening was carried out by DHPLC and restriction fragment analysis. The usage of Optimase Taq polymerase results in complete reversion of the first and second mutated nucleotide introduced at the 3' end of the mutagenic reverse primer. It also partially reverses the missense nucleotide introduced in the third position of the mutagenic primer and leads to misleading DHPLC and restriction fragment analysis patterns. Nevertheless it cannot perform such an activity when an abnormal nucleotide is introduced in the fourth position.     

‘Petite’ mutagenesis and mitotic crossing-over in yeast by DNA-targeted alkylating agents

Although the biological properties (cytotoxicity, mutagenicity and carcinogenicity) of alkylating agents result from their bonding interactions with DNA, such compounds generally do not show any special binding affinity for DNA. A series of acridine-linked aniline mustards of widely-varying alkylator reactivity have been designed as DNA-directed alkylating agents. We have considered whether such DNA targeting has an effect on mutagenic properties by evaluating this series of drugs in comparison with their untargeted counterparts for toxic, recombinogenic and mutagenic properties in Saccharomyces cerevisae strain D5. The simple untargeted aniline mustards are effective inducers of mitotic crossing-over in this strain, but resemble other reported alkylators in being rather inefficient inducers of the “petite” or mitochondrial mutation in yeast. However, the majority of the DNA-targeted mustards were very efficient petite, mutagens, while showing little evidence of mitotic crossing-over or other nuclear events. The 100% conversion of cells into petites and the lack of a differential between growing and non-growing cells are similar to the effects of the well characterised mitochondrial mutagen ethidium bromide. These data suggest very different modes of action between the DNA-targeted alkylators and their non-targeted counterparts.     

Modulation of mutagenic properties in a series of DNA-directed alkylating agents by variation of chain length and alkylator reactivity.

Four series of aniline mustards linked to a DNA-affinic acridine chromophore by alkyl chains of varying length (2-5 carbon atoms) have been studied for their mutagenic properties, as estimated in four strains of Salmonella typhimurium and in Saccharomyces cerevisiae strain D5. The four series have very different mustard reactivities, as determined by the aniline link group (-O-, -CH2-, -S- or -SO2-). Some of the derived compounds cause frameshift mutagenesis which can be detected in TA98 and also "petite" mutagenesis activity, neither of which occur to significant extents with the parent mustards or with 9-aminoacridine. None of the derived compounds are as effective as the parent mustards in mitotic crossing-over, nor do they show ability for frameshift mutagenesis in S. typhimurium TA1977 which is typical of acridines. Some of the compounds have comparable frameshift activity to compounds such as ICR-191, but appear to have a different base-pair preference. The results indicate clear structure-activity relationships for the spectrum of mutagenic activity, which relate to both chain length and alkylator reactivity, for these compounds.     

Molecular cloning and partial characterization of the pathway for aniline degradation inPseudomonas sp. strain CIT1

The genes encoding aniline utilization inPseudomonas sp. strain CIT1 have been cloned inEscherichia coli and partially characterized. Molecular cloning of the genes was achieved by construction of a cosmid library, followed by mobilization of the library into mutants ofPs. sp. CIT1 impaired in a number of functions necessary for growth on aniline. A 42-kbSau3A fragment was found to encode the ability to utilize aniline and contained the catechol 2,3-dioxygenase (C230) gene. The regions encoding these activities were subcloned and further characterized. Plasmids containing the aniline oxidase gene encoded a 260-kDa protein complex, which was putatively shown to be composed of 72 kDa and possibly 36 kDa subunits. The fragment required for C230 activity encodes a 35 kDa protein, similar in size to C230 gene products previously characterized.     

An investigation of the metabolism of N-nitroso-N-methylaniline by phenobarbital- and pyrazole-induced Sprague-Dawley rat liver and esophagus derived S-9

The metabolism and mutagenicity of the esophageal carcinogen N-nitroso-N-methylaniline (NMA) was studied using hepatic and esophageal 9000 X g supernatant (S-9) preparations from Sprague-Dawley rats induced with pyrazole and phenobarbital. Only pyrazole-induced hepatic S-9 was able to dose-dependently activate NMA to a mutagen in the Ames assay and specifically in Salmonella typhimurium TA1537. NMA in the presence of phenobarbital-induced S-9 gave a very weak non-dose dependent mutagenic response. Metabolism of NMA by the two induced hepatic and esophageal S-9 fractions yielded aniline and N-methylaniline (MA). Phenobarbital-induced S-9 from both tissues also afforded phenol, while none was found with the pyrazole-induced preparations. Phenol formation presumably arose from the direct oxidative demethylation of NMA via a benzenediazonium ion (BDI) intermediate. The results indicate that an important metabolic pathway for NMA, with both inducing agents, entails an initial denitrosation to yield MA, which in turn rapidly undergoes oxidative demethylation to aniline. The conversion of NMA to phenol also suggests that direct demethylation of NMA in the phenobarbital-induced system is an important metabolic pathway.     

Role of the oncogene in the mutagenic activity of bovine adenovirus type 3

The mutagenic and carcinogenic effect of two EcoRI-fragments of bovine adenovirus type 3 (BAV-3) DNA inserted into pBR325 has been studied. The C fragment (located between 3,6 and 19,7 map units) contains the viral oncogene, the C fragment (between 44,3 and 63,7 map units) displays no transforming activity. It has been established that oncogene BAV-3 statistically true increases the yield of mutants resistant to 6-mercaptopurine (6MP) in Chinese hamster cells. The C fragment, pBR325 without viral sequences and DNA fragments of different molecular weights from normal Syrian hamster cells have no mutagenic effect. The control over tumor formation in syngenic mice after injection of C3H10T 1/2 and D. C fragments and pBR325 treatment exposed a parallelism between the mutagenic and transforming effect. The study of the combined effect of viral DNA fragments and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) which increases the transforming activity of different carcinogens, shows that the promoter increases the frequency of mutants after viral oncogene treatment and does not induce mutagenic activity of those types of DNA which are unable to transform the cells.     

Design and synthesis of potent carboxylic acid DGAT1 inhibitors with high cell permeability

A series of potent carboxylic acid DGAT1 inhibitors with high permeability were developed from a virtual screening hit. Strategies were employed to reduce Pgp substrate recognition and increase passive permeability, resulting in the discovery of a series showing good inhibition of cellular triglyceride synthesis. The mutagenic potential of prospective metabolites was evaluated in the Ames assay, and one aniline was shown to be devoid of mutagenicity.     

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.     

Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA 1538

A series of ten azo dyes as well as various single ring aromatic amines substituted on the benzene ring were tested for bacterial mutagenicity with Salmonella typhimurium TA 1538 using a soft-agar overlay method. Two dyes, sudan 2 and chrysoidin induced mutation but only in the presence of a rat liver preparation. Chrysoidin was the more active. Testing of its reduction products, aniline and 1,2,4-triaminobenzene showed a liver metabolite of the latter compound could be responsible for the mutagenic effect, having a comparable mutagenicity with 1,2-diamino-4-nitro-benzene, one of the mutagenic constituents of hair dyes. Structure-activity studies on a series of ring-substituted anilines indicated that mutagenic activity required at least two positions to be substituted with either amino or nitro groups, or one of each. The bacteria as well as the liver enzyme preparation may partake in the activation of these chemicals. The correlation between mutagenicity and carcinogenicity for this group of compounds is discussed.     

Stimulation of mutations suppressing the loss of replication control by small alcohols

Transient exposure of lysogenic Escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. The stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propanol to evoke mutagenic effects. The selected mutant cells were, in general, equally or more sensitive to ethanol than the starting cells. The mutagenicity of methanol and ethanol was detected only with E. coli strains with lambda fragments that included the site-specific and general recombination genes found within the phage int-kil gene interval; whereas, stimulation of the frequency of phenotypically identical mutations by nitrosoguanidine or ionizing radiation did not require that the lambda fragment encode these genes. Treatments of lysogenic cells with mutagenic concentrations of ethanol did not trigger prophage induction and were concluded not to induce a cellular SOS response nor to denature the prophage repressor, or to disrupt repressor-operator binding. The toxicity of ethanol was pH-dependent. Cellular sensitivity to ethanol toxicity was unaffected by the integrated lambda fragment(s) or by an intact lambda prophage; but, it was increased by deletions of the E. coli chromosome extending rightward from bio into uvrB, and rightward from chlA.     

Mutagenicity testing of some commonly used dyes.

Seventeen commonly used dyes and 16 of their metabolites or derivatives were tested in the Salmonella-mammalian microsome mutagenicity test. Mutagens active with and without added Aroclor-induced rat liver microsome preparations (S9) were 3-aminopyrene, lithol red, methylene blue (USP), methyl yellow, neutral red, and phenol red. Those mutagenic only with S9 activation were 4-aminopyrazolone, 2,4-dimethylaniline, N,N-dimethyl-p-phenylenediamine, methyl red, and 4-phenyl-azo-1-naphthylamine. Orange II was mutagenic only without added S9. Nonmutagenic azo dyes were allura red, amaranth, ponceau R, ponceau SX, sunset yellow, and tartrazine. Miscellaneous dyes not mutagenic were methyl green, methyl violet 2B, and nigrosin. Metabolites of the azo dyes that were not mutagenic were 1-amino-2-naphthol hydrochloride, aniline, anthranilic acid, cresidine salt, pyrazolone T,R-amino salt (1-amino-2-naphthol-3,6-disulfonic disodium salt), R-salt, Schaeffer's salt (2-naphthol-6-sulfonic acid, sodium salt), sodium naphthionate, sulfanilamide, and sulfanilic acid. 4-Amino-1-naphthalenesulfonic acid sodium salt was also not mutagenic. Fusobacterium sp. 2 could reductively cleave methyl yellow to N,N-dimethyl-p-phenylenediamine which was then activated to a mutagen.     

Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells treated with aminophenylnorharman formed by norharman with aniline

Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix. Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix. Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix. In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH. On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH. The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH. Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells. A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH. The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb). In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature. Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.     

Genetic diversity among 3-chloroaniline- and aniline-degrading strains of the Comamonadaceae

We examined the diversity of the plasmids and of the gene tdnQ, involved in the oxidative deamination of aniline, in five bacterial strains that are able to metabolize both aniline and 3-chloroaniline (3-CA). Three strains have been described and identified previously, i.e., Comamonas testosteroni I2 and Delftia acidovorans CA28 and BN3.1. Strains LME1 and B8c were isolated in this study from linuron-treated soil and from a wastewater treatment plant, respectively, and were both identified as D. acidovorans. Both Delftia and Comamonas belong to the family Comamonadaceae. All five strains possess a large plasmid of ca. 100 kb, but the plasmids from only four strains could be transferred to a recipient strain by selection on aniline or 3-CA as a sole source of carbon and/or nitrogen. Plasmid transfer experiments and Southern hybridization revealed that the plasmid of strain I2 was responsible for total aniline but not 3-CA degradation, while the plasmids of strains LME1 and B8c were responsible only for the oxidative deamination of aniline. Several transconjugant clones that had received the plasmid from strain CA28 showed different degradative capacities: all transconjugants could use aniline as a nitrogen source, while only some of the transconjugants could deaminate 3-CA. For all four plasmids, the IS1071 insertion sequence of Tn5271 was found to be located on a 1.4-kb restriction fragment, which also hybridized with the tdnQ probe. This result suggests the involvement of this insertion sequence element in the dissemination of aniline degradation genes in the environment. By use of specific primers for the tdnQ gene from Pseudomonas putida UCC22, the diversity of the PCR-amplified fragments in the five strains was examined by denaturing gradient gel electrophoresis (DGGE). With DGGE, three different clusters of the tdnQ fragment could be distinguished. Sequencing data showed that the tdnQ sequences of I2, LME1, B8c, and CA28 were very closely related, while the tdnQ sequences of BN3.1 and P. putida UCC22 were only about 83% identical to the other sequences. Northern hybridization revealed that the tdnQ gene is transcribed only in the presence of aniline and not when only 3-CA is present.     

Genotoxicity of aniline derivatives in various short-term tests

Various substituted aniline derivatives were tested for genotoxicity in several short-term tests in order to examine the hypothesis that a substitution at both ortho positions (2,6-disubstitution) could prevent genotoxicity due to steric hindrance of an enzymatic activation to electrophilic intermediates. In the Salmonella/microsome assay, 2,6-dialkylsubstituted anilines and 2,4,6-trimethylaniline (2,4,6-TMA) were weakly mutagenic in strain TA100 when 20% S9 mix was used, although effects were small compared to those of 2,4-dimethylaniline and 2,4,5-trimethylaniline (2,4,5-TMA). In Drosophila melanogaster, however, 2,4,6-TMA and 2,4,6-trichloroaniline (TCA) were mutagenic in the wing spot test at 2-3 times lower doses than 2,4,5-TMA. In the 6-thioguanine resistance test in cultured fibroblasts, 2,4,6-TMA was again mutagenic at lower doses than 2,4,5-TMA. Two methylene-bis-aniline derivatives were also tested with the above methods: 4,4'-methylene-bis-(2-chloroaniline) (MOCA) was moderately genotoxic in all 3 test systems whereas 4,4'-methylene-bis-(2-ethyl-6-methylaniline) (MMEA) showed no genotoxicity at all. DNA binding studies in rats, however, revealed that both MOCA and MMEA produced DNA adducts in the liver at levels typically found for moderately strong genotoxic carcinogens. These results indicate that the predictive value of the in vitro test systems and particularly the Salmonella/microsome assay is inadequate to detect genotoxicity in aromatic amines. Genotoxicity seems to be a general property of aniline derivatives and does not seem to be greatly influenced by substitution at both ortho positions.