Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene.

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.

Synthesis of inducible enzymes in irradiated yeast cells. Ultraviolet effects on transcription and the influence of recovery processes.

The induced synthesis of arginase was measured in several yeast strains after ultraviolet light irradiation. There was an exponential dose-related reduction which was the same in all cell lines tested. This sensitivity is compatible with the size of the arginase structural gene if it is assumed that one pyrimidine dimer suffices for blocking transcription. The ultraviolet-induced synthesis inhibition is susceptible to photoreactivation and liquid holding recovery. The latter process is absent in a rad2 mutant and reduced in a rad9 mutant.

Expression of radiation-induced mutations at the arginine permease (CAN1) locus in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the expression of resistance to the L-arginine analog, L-canavanine, after mutagenesis, is strongly dependent on the metabolic state of the cell. The frequency of mutations recovered after exposure to ultraviolet light or X rays was measured under a variety of culture conditions. The results indicate that the frequency of mutants recovered is determined by the following three factors: (1) The potential mutants still possess enough permease activity to take up some of the cell poison, and some are therefore killed before they can express the mutant genotype. The sensitivity is strongly influenced by the endogenous free arginine, which is in turn influenced by the growth medium. (2) The rapid decay of the permease molecules and the inability of the potential mutants to resynthesize this protein results in a rapidly increasing change of expression when selection is delayed. (3) During the time when the permease activity is decaying, repair of the mutagen-induced damage appears to occur.

The CAN1 locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates.

A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.

Plasmid copy number and mutant frequencies in S. typhimurium TA102.

Tetracycline and chloramphenicol increase the number of mutant colonies of strain TA102, which carries the reverting gene on the plasmid pAQ1. Determination of the plasmid content by agarose gel analysis shows that the increase of the mutant colony number is paralleled closely by an increase of the number of pAQ1 plasmids per cell, indicating that the two compounds do not increase the frequency of mutants "per gene," but only enhance the number of the genes at which mutations can occur. Thus, not considering the molecular processes could result in mistakenly attributing the increase in the number of mutants per plate (respective to the number of mutants per cell) to a mutagenic activity of the antibiotics.

Phenobarbital: does the positive result in TA1535 indicate genotoxic properties?

The liver carcinogen phenobarbital (PB) causes a weak but reproducible increase of the mutant frequency in the Ames test, strain TA1535, without S9. Since there is no obvious chemical basis for a "DNA reactivity" of this compound experiments were performed to obtain information about possible indirect mechanisms of enhancing the number of spontaneous mutant colonies. In the course of the study strong synergistic and comutagenic effects of PB when given in combination with Na-azide or 2-aminoanthracene (2AA) were observed. Not only TA1535 but the complete set of tester strains was responsive. However, PB did not enhance the effects of other mutagens such as 4-nitroquinoline N-oxide or 2-nitrofluorene. It is argued that in strain TA1535 the fixation and expression of spontaneously occurring DNA lesions is amenable to modulation by PB similar to that of Na-azide or 2AA induced lesions. Thus in the usual sense, PB is not genotoxic in the Ames test. Methapyrilene, another liver carcinogen with an assumed nongenotoxic mode of action, showed almost identical properties in these experiments.

Evaluation of the micronucleus test in vitro using Chinese hamster cells: results of four chemicals weakly positive in the in vivo micronucleus test.

A rapid and simple procedure for the micronucleus test (MNT) in vitro using Chinese hamster ovary (CHO) cells was established in our laboratory. The assay is intended to quickly screen chromosomal aberrations in vitro within the framework of industrial genotoxicity studies. To test the sensitivity of the assay in the experiments described here, four substances, classified as noncarcinogens but reported as weak inducers of micronuclei (MN) in bone-marrow cells of mice, were evaluated in the MNT in vitro. Of the four compounds, ascorbic acid, phenol, and 2,6-diaminotoluene proved to be genotoxic in the MNT in vitro. Titanium dioxide, which could not be dissolved in the culture medium, did not induce MN. The MNT in vitro proved to be quick and relatively simple and to yield highly reproducible results when testing the four chemicals.

Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group.

Recent toxicological observations have caused concern regarding the need to test, for example, pharmaceuticals and cosmetic products for photochemical genotoxicity. The objective of this report is to give assistance on how to adapt existing test methods to investigate the potential of light-absorbing compounds to induce genotoxic effects on photoactivation. In general, the Organization for Economic Co-Operation & Economic Development (OECD) draft guideline on in vitro phototoxicity testing served as a basis for consideration. Concomitant exposure of the cells to the test compound and solar simulated light was considered appropriate as the initial, basic test condition. Optimization of the exposure scheme, e.g., a change of the irradiation spectrum, might be indicated depending on the initial test results. Selection of test compound concentrations should be based on results obtained with the dark version of the respective test system but might have to be modified if phototoxic effects are observed. Selection of the irradiation dose has to be performed individually for each test system based on dose-effect studies. The irradiation should induce per se a small, reproducible toxic or genotoxic effect. The report includes a specification of necessary controls, discusses factors that might have an impact on the irradiation characteristics, and gives a rationale for the omission of an external metabolic activation system. It also addresses the question that physicochemical and pharmacokinetic properties might trigger the need to test a chemical for photochemical genotoxicity. Relevant experimental observations are presented to back up the recommendations. The working group did not reach a consensus as to whether a single, adequately perfomed in vitro test for clastogenicity would be sufficient to exclude a photogenotoxic liability or whether a test battery including a gene mutation assay would be needed for product safety testing regarding photochemical genotoxicity.

Mutagenicity study of Remsen-Fahlberg saccharin and contaminants.

Saccharin and contaminants of commercial Remsen-Fahlberg saccharin were studied for mutagenic potential with the use of the Salmonella/microsome test, Basc-test in Drosophila melanogaster and micronucleus test in mice. In none of these tests were mutagenic effects of saccharin observed. Likewise, the ortho- and para-sulfamoylbenzoic acids (OSBA and PSBA) were ineffective. Para-toluenesulfonamide (PTS) and the major contaminant ortho-toluene-sulfonamide (OTS) exhibited weak mutagenic effects in a modified Salmonella/microsome test and in Drosophila. These results do not indicate mutagenic and therewith correlated carcinogenic potential of saccharin, but they emphasize the possible activity of contaminants.

The photomutagenicity of fluoroquinolones and other drugs.

Induction of DNA damage as a consequence of exposure to UV light has been established as the major and still increasing cause of skin cancer. Absorption of the photon energy may be either directly by the DNA molecules (for wavelengths < 320 nm) or may be by endogenous or exogenous chemicals (sensitizers) with the potential of energy or electron transfer to DNA. Oxygen-mediated reactions (often called type II reactions) appear to be the most important mechanism since molecular oxygen is a good and abundant substrate for triplet excited sensitizers. Energy transfer to molecular oxygen is possible for wavelengths in the near UV and in the visible part of the solar spectrum since the energy of the excited oxygen molecule ((1)O2*) is comparatively low. A few light-absorbing pharmaceuticals have long been known to cause photo(geno)toxic effects. Notably psoralene and chlorpromazine derivatives have been established as photomutagens and the reaction mechanisms have been identified. The fluoroquinolone antibiotics have more recently been recognized as being photomutagenic. The type of DNA damage and the modulation by antioxidants indicate the involvement of reactive oxygen species (ROS) but other mechanisms are also reported at least for some derivatives. In routine genotoxicity studies we observed a photomutagenic activity of a compound under development as an anxiolytic agent in the Ames tester strain TA102 at 'normal laboratory illumination' conditions. Further investigations showed strong photogenotoxic activity in tests for gene mutations and chromosomal aberrations in mammalian cells. The compound proved to be a potent (1)O2-producer. The finding led to termination of development but in the course of studies several structural analogues have been tested for which structure activity relationships will be described. The relevance of photogenotoxic properties of drugs for predicting adverse effects in man will be discussed.

Mechanism of quinolone mutagenicity in bacteria.

Gyrase inhibitors of the quinolone type are genotoxic in bacteria. A functioning excision-repair system is needed to show the mutagenic activity in the Ames tester strains. The antibiotics do not interact with DNA directly but induce the gyrase enzyme to cleave the DNA with protein covalently bound at the site-specific double-strand scission. A prominent site of action is shown to be located at a distance of about 80 bp from the hisG428 sequence at which mutations are scored in the responsive strain TA102. Since the interaction of quinolones with the mammalian counterparts of gyrase, topoisomerase II, is lower by orders of magnitude, it is argued that the bacterial screening tests are of little relevance for predicting effects in mammalian cells.

Reduction of the translation fidelity by kanamycin: effects on growth and mutant frequency in S. typhimurium TA102.

Kanamycin reduces the translation fidelity in prokaryotes. By read-through of the ochre stop codon (hisG428) of S. typhimurium TA102 enough functional enzyme is produced to allow the his- cells to form a dense background growth on the minimal agar plates. The influence on the revertant colony numbers is similar to the effect of histidine supplementation.

Review of the genotoxic properties of chlorpromazine and related phenothiazines.

Chlorpromazine and related phenothiazine drugs have been used in human and veterinary medications for more than 40 years, predominantly as psychotropic agents. Genotoxicity reports are in many cases of relatively antiquated test design. Overall there appears to be no genotoxic activity associated with these drugs when tested under standard conditions. Limited evidence for the potential to form mutagenic nitrosation products and some indication for the ability to modulate the genotoxic action of various mutagens have been presented in the literature. UV irradiation of chlorpromazine and other chlorinated derivatives produces reactive free radicals which possess DNA damaging properties. Induction of gene mutation and chromosomal aberrations have been observed in appropriately designed photomutagenesis experiments. Enhancement but also reduction of UV induced skin tumour formation by chlorpromazine have been found. The decisive factor for the discrepant actions has not been recognized. It is clearly advisable to avoid extensive UV exposure during therapy with these drugs.

The 'antimutagenic' effect of cinnamaldehyde is due to a transient growth inhibition.

Addition of cinnamaldehyde to the selective medium causes a reduction in the number of revertant colonies of S. typhimurium or E. coli when the cells have been mutagenized with 4NQO but not when they have been mutagenized with MNNG. Toxicity of the cinnamaldehyde exposure depends largely on the status of growth and/or nutrient supply of the cells. We present evidence that simple growth inhibition due to lack of nutrients mimics the effect of cinnamaldehyde in 4NQO- and MNNG-treated cells. This argues that the reduction of mutant colonies is due to a transient growth retardation caused by cinnamaldehyde exposure, which presumably allows the cells to repair 4NQO-induced damage--but not MNNG-induced damage--via a more error-free pathway.

Mutagenicity testing experiments with the Cobas Bact.

The feasibility of mutagenicity assays with the Cobas Bact Automatic analyser was explored using selected model mutagens. The reduction of the latency period (the period until the growth of the mutant cells becomes optically measurable) was found to be a valid measure for the mutagenic activity of strong mutagens. For weaker mutagens an evaluation analogous to the fluctuation test seemed the more appropriate approach. The influence of various variables, such as concentration of histidine, size of inoculum, medium composition and S9 concentration, is described. Adaptation of the Cobas Bact system to the differential growth inhibition test is also mentioned.

Renin inhibitors as an example of presumptive irrelevant positive findings in the Salmonella/mammalian microsome assay (Ames test).

An increase in the number of mutant colonies in the Ames test is generally taken as a strong indication for a genotoxic (e.g., DNA damaging) property of the test compound or its metabolites. However, a few examples are known in which mechanisms usually related to some sort of growth enhancement will lead to increases in mutant frequencies of spontaneous origin. The renin inhibitor Ro 42-5892 increased the number of mutant colonies of strain TA1538 and to a lesser degree of TA98 in the standard plate incorporation assay (Ames test). Since there is no chemical basis for a 'DNA reactivity' of this compound, experiments were performed to obtain information about possible indirect mechanisms of enhancing the number of spontaneous mutant colonies. Circumstantial evidence is presented to attribute the weak activity not to an inherent genotoxic property but rather to an as yet undefined indirect effect on the expression of spontaneous mutants. Since Ro 42-5892 contains a histidine residue it was a reasonable assumption to suspect a growth enhancing property of the test compound. However, none of the strains showed an elevation of the number of revertant colonies or an increase in the density of the background growth. In addition, structurally related non-histidine containing renin inhibitors showed absolutely no increase in the number of revertant colonies. Furthermore, no growth induction (either in liquid or under selective conditions) and no histidine cleave off by a TA1538/TA98 specific metabolism could be shown. A second line of evidence showing parallelism to growth enhancing compounds concerns the comutagenicity of histidine containing renin inhibitors. When Ro 42-5892 was tested in combination with established mutagens, a multiplicative synergism was found. This effect was observed not only in strains TA1538 and TA98 but also in the standard Salmonella tester strains where the spontaneous mutant frequency was not increased by Ro 42-5892. Analogous effects were previously shown for free histidine, isohistidine, phenobarbital and tetracycline and in part explained by molecular mechanisms.

Synergistic/comutagenic action in the Ames test as an indication of irrelevant positive findings.

A number of structurally very diverse compounds which cause weak positive effects in the Ames test by evident or suspect irrelevant mechanisms is discussed. As a unifying observation we describe synergistic effects in combination with known mutagens in the responsive strains and comutagenic effects in initially unresponsive strains. We argue that the compounds enhance the formation of spontaneous (or mutagen-induced) revertant colonies by test-specific mechanisms likely to be of no relevance to multicellular eukaryotic organisms rather than possessing intrinsic genotoxic (i.e. DNa-damaging) properties in the Ames test.

Photomutagenesis test development: II. 8-Methoxypsoralen, chlorpromazine and sunscreen compounds in chromosomal aberration assays using CHO cells.

Chromosomal changes were analysed in Chinese hamster ovary (CHO) cells treated with 8-methoxypsoralen (8-MOP) or chlorpromazine (CPZ) and irradiated with either a UVA fluorescent tube (emission spectrum ranging from 350 to 400 nm) or a xenon burner (continuous emission spectrum simulating ambient sunlight). In the dark neither 8-MOP nor CPZ was genotoxic by itself. If these compounds were used in combination with UV irradiation the rate of chromosome aberrations was significantly increased. The magnitude of the clastogenic response was dependent on compound concentration and UV dose. The spectral composition also played an important role. Care must be taken to account for spectral changes caused, e.g., by passage of the light through the plastic lid of the container. The possible clastogenicity of two sunscreens was tested with two protocols: (1) cells attached to the culture dish were treated in presence of the sunscreen in the medium or (2) cells were irradiated through a layer of sunscreen solution as a filter. With this a clear UVB-absorbing effect and a decreased frequency of UVAB-induced chromosome aberration was evident with the UVB-absorbing compound Parsol HS but was absent, as expected, with the UVA-absorbing compound Parsol 1789. The presence of the sunscreens in the irradiated cell sample did not cause a significant increase in UV-induced chromosome aberrations.

Mutation induction and mutation spectra of S. typhimurium TA100 after exposure to isohistidines.

L-Isohistidine and D,L-isohistidine, but not D-isohistidine, caused an increase of the number of mutant colonies in S. typhimurium strain TA100. Spontaneous and also sodium azide or 2-aminoanthracene induced mutant numbers were enhanced by L-isohistidine and by an isomeric mixture of D,L- and L-isohistidine. These effects could not be attributed to a growth-enhancing property. The colony probe hybridization procedure was used to investigate the effects of the histidines on the spontaneous and azide-induced spectra of the hisG46 allele in strain TA100. D,L-Isohistidine, but not the D-isomer, caused and increase of transitions (CCC-->CTC) and transversions (CCC-->CAC) in the spontaneous spectrum. Sodium azide alone induced a strong increase of CCC-->CTC transitions; combination with the D,L-isohistidine led to a further enhancement of this type of base substitutions, whereas with the L-isomer, no such effect was observed. This supports the hypothesis that the activity of D,L-isohistidine is probably not due to DNA-damaging properties, but rather to indirect mechanisms, such as enhancement of the infidelity of DNA replication and/or interference with DNA-repair or proofreading functions.

Photomutagenesis test development: I. 8-Methoxypsoralen, chlorpromazine and sunscreen compounds in bacterial and yeast assays.

Two in vitro genotoxicity tests have been adapted to the evaluation of photomutagenic activity of test compounds. The study was initiated to obtain an experimental basis relating to newly proposed guidelines of the EC which request the screening of UV-absorbing compounds, for example, those employed in sunscreen preparations, for their photomutagenic potential. The well established photomutagens 8-methoxypsoralen and chlorpromazine were used to define relevant test protocols. The compounds were evaluated with the Ames test and the Saccharomyces cerevisiae D7 test for gene conversion. The influence of various parameters such as UV light sources, spectral composition, UV sensitivity of the test systems, absorbance by test materials and different exposure conditions is indicated. Two exemplary screening experiments with cosmetic ingredients are presented. Both test systems can be employed for the evaluation of compounds for photomutagenic activity although the standard excision-deficient strains of S. typhimurium pose problems because of their high UV sensitivity. The present experience in this complex field suggests that rigid test protocols and a restrictive test battery would be inadequate.