Showing papers in "Mutation Research in 1996"

Smoking and low antioxidant levels increase oxidative damage to sperm DNA.

Abstract: Our previous studies have shown that men with low ascorbate intake have markedly increased oxo8dG in the DNA of their sperm. Because cigarette smoke is high in oxidants and depletes plasma and tissue antioxidants, oxidative DNA damage in sperm and tocopherol and ascorbate levels in seminal plasma were determined in smokers and non-smokers. The level in sperm DNA of oxo8dG, an oxidative lesion of guanine, was 50% higher in smokers compared to nonsmokers (p = 0.005). The concentration of alpha-tocopherol in seminal plasma was decreased in smokers by 32% (p = 0.03). Smoking and low antioxidant levels increase oxidative damage to sperm DNA. We discuss the possibility that paternal smoking causes mutations in sperm that lead to cancer, birth defects, and genetic diseases in offspring.

DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes

Abstract: Bleomycin and the enediyne antibiotics effect concerted, simultaneous site-specific free radical attack on sugar moieties in both strands of DNA, resulting in double-strand breaks of defined geometry and chemical structure, as well as abasic sites with closely opposed strand breaks. The hypersensitivity of several mammalian double-strand break repair-deficient mutants to these agents confirms the role of these double-strand breaks in mediating cytotoxicity. In bacteria, mutagenesis by both bleomycin and neocarzinostatin appears to result from replicative bypass of abasic sites, the repair of which is blocked by the presence of closely opposed strand breaks. However, in mammalian cells, such abasic sites decompose to form double-strand breaks, and mutagenesis consists primarily of small deletions, large deletions, and gene rearrangements, all of which probably result from errors in double-strand break repair by a nonhomologous end-joining mechanism. Studies with the radiomimetic antibiotics emphasize the importance of this end-joining repair pathway, and these agents provide useful probes of its mechanistic details, particularly the effects of chemically modified DNA termini on repair.

DNA adducts from chemotherapeutic agents

Abstract: The guiding principle of early work was the hypothesis that the anti-cancer alkylating drugs acted through their ability to cross-link macromolecules essential for cell division. Not long afterwards, DNA was specified as the essential target, and support for the hypothesis came from evidence that the archetypal agent, mustard gas, could link guanine bases in DNA through their N-7 atoms. Quantitative correlations between alkylation of DNA and its inactivation as a template followed, with bacteriophage as a simple test object, showing that the mean lethal dose was close to a single cross-link in the genome. This conclusion applied to either mustard gas or the more recently introduced platinum drugs. Although both inter- and intra-strand cross-links were effective, it was thought that in cells the inter-strand cross-link would, by preventing the separation of the strands necessary for cell division, and by being more difficult to repair, constitute the more effectively lethal lesion. With repair-deficient bacteria, it also emerged that a single cross-link in the genome was lethal, but proficient bacteria could remove about 20 cross-links through excision repair. Mono-7-alkylguanines were not removed and were evidently inert. Thus, only a few percent of the total alkylation products were the most effective lesions. Parallel studies with cultured mammalian cells gave a rather different picture, in that the mean lethal doses of even hypersensitive cell lines were around 20 or more cross-links per genome, about the same as for resistant strains of bacteria. Most cells could withstand several hundreds of cross-links per genome, and although adducts were removed, there was incomplete removal of cross-links. Some, but not all, sensitive cell lines were deficient in excision repair. Methods were devised for measuring the extents of alkylation of DNA in cells of patients treated with chemotherapeutic drugs; these are mainly immunoassays, and were applied generally to peripheral blood leukocytes, although some tumours were studied. Extents of alkylation of leukocyte DNA were generally of the same order as, or rather less than the mean lethal doses of cultured cells of the 'normal' type, but in some reports for cisplatin-treated patients, very wide variability between individuals was found. A positive correlation between adduct levels, and particularly a very minor adduct recognised specifically by one antibody, and favourable therapeutic outcome was discerned, and suggested to have a pharmacogenetic basis. In several instances, extents of alkylation of tumours were significantly higher than the average for leukocytes; for ovarian and a testicular tumour for cisplatin, and for a plasma cell tumour for melphalan. Nevertheless, these favourable examples would not constitute more than three or four mean lethal doses in the tumour cells, assuming that they had the same sensitivity as 'normal' cell lines: the therapeutic effect would of course be much more favourable if the tumour cells resembled 'sensitive' cell lines. This lack of a favourable difference between extents of alkylation in DNA of patients and the mean lethal dose for normal cells was particularly obvious with the methylating drugs dacarbazine and procarbazine. These considerations stress the need for higher extents of alkylation to be achieved in target tumour DNA for successful chemotherapy. One approach is to give a higher overall dose, and to 'rescue' the bone marrow (known from the earliest report on mustard gas to be the most susceptible tissue) by autologous transplantation. The second, which has yet to reach the clinic, is to convert unreactive prodrugs through enzymic activation into alkylating agents specifically in tumours (see Bagshawe, 1994).

Radioprotective effects of antioxidative plant flavonoids in mice

Abstract: Radioprotective effects of tea infusions and plant flavonoids were investigated by using the micronucleus test for anticlastogenic activity and the thiobarbituric acid assay for antioxidative activity. A single gastric intubation of rooibos tea (Aspalathus linearis) infusion at 1 ml per mouse 2 h prior to gama-ray irradiation (1.5 Gy) reduced the frequency of micronucleated reticulocytes (MNRETs). After the fractionation of rooibos tea infusion, the flavonoid fraction was found to be most anticlastogenic and antioxidative. From this fraction, luteolin was isolated as an effective component. Then, anticlastogenic effects of 12 flavonoids containing luteolin and their antioxidative activities against lipid peroxidation by Fenton's reagent were examined. A good correlation (r=0.717) was observed between both activities. Luteolin showed the most effective potency. A gastric intubation of luteolin (10 micromoles/kg) 2 h prior to gamma-ray irradiation (6 Gy) suppressed lipid peroxidation in mouse bone marrow and spleen and a trend of protective effect of luteolin against the decrease of endogenous ascorbic acid in mouse bone marrow after gamma-ray irradiation (3 Gy) was observed. These results suggest that plant flavonoids, which show antioxidative potency in vitro, work as antioxidants in vivo and their radioprotective effects may be attributed to their scavenging potency towards free radicals such as hydroxyl radicals. Therefore, the flavonoids contained in tea, vegetables and fruits seem to be important as antioxidants in the human diet.

Hypersensitivity to very-low single radiation doses: Its relationship to the adaptive response and induced radioresistance

Abstract: There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

Abstract: To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.

Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells

Abstract: Peroxynitrite is a powerful oxidant formed through reaction of nitric oxide with superoxide. Because activated macrophages can produce both nitric oxide and superoxide, it has been proposed that peroxynitrite may contribute to cytotoxicity and increased cancer risks associated with the inflammatory response during chronic infections. We therefore investigated mutagenicity of peroxynitrite in the supF gene of the pSP189 shuttle vector as a mutation target. The plasmid was exposed to 2.5 mM peroxynitrite in vitro, then replicated in Eschericia coli MBL50 and in human AD293 cells. Mutation frequency increased 21-fold in pSP189 replicated in E. coli and 9-fold in plasmid replicated in human cells. Mutations were clustered within the 5' region of the supF gene in plasmids replicated in bacteria. The hot spots were located at positions 108, 113, 116, 124, 126 and 141; more than 25% of all mutations occurred at position 124. Following replication in human cells, mutations were more widely distributed over the gene, with hot spots at positions 113, 124, 133, 156 and 164; 15% occurred at position 124. In both systems, the majority of mutations occurred at G:C base pairs, predominantly involving G:C-->T:A transversions (65% when replication was in bacteria and 63% when in human cells). G:C-->C:G transversions were observed at lower frequency (28% in MBL50 and 11% in AD293 cells), and 11% of mutations found in vectors replicated in AD293 cells were G:C-->A:T transitions. A greater number of large deletions, insertions, tandem and multiple mutations occurred in plasmid replicated in AD293 cells. Differences in mutation spectra following replication in the two systems may be attributable to differences in recognition and repair of the lesions and/or properties of the replication apparatus.

Cadmium is an inducer of oxidative stress in yeast

Abstract: The heavy metal cadmium is a carcinogen in long-term rodent studies and is a suspect human carcinogen. Cadmium scores negative in the Ames Salmonella mutagenicity assay and in most other short-term genotoxicity assays, but induces deletions in the yeast Saccharomyces cerevisiae. We have investigated whether cadmium induces an oxidative stress in S. cerevisiae which may be responsible for its recombinagenic activity. The free radical scavenger N-acetylcysteine blocked toxicity and recombination induced in S. cerevisiae by cadmium. Yeast strains deficient in the antioxidant defense enzymes superoxide dismutase or gamma-glutamylcysteine synthetase were hypersensitive to cadmium toxicity. Cells grown in the absence of oxygen were more resistant to cadmium. An intracellular free radical-sensitive reporter compound was activated in S. cerevisiae exposed to cadmium. Toxicity or recombination induced by the mutagenic carcinogen methyl methanesulfonate were unaffected in any of the above experiments. These results suggest that the toxicity and recombinagenic activity of cadmium in S. cerevisiae is caused by oxidative stress.

Aspects of the adaptive response to very low doses of radiation and other agents

Abstract: When human lymphocytes and other cells are pre-exposed to very low doses of ionizing radiation and subsequently exposed to a high dose, less genetic damage, i.e., fewer chromosome aberrations, is found than is observed in cells that had not been pre-exposed. This has been termed the adaptive response and has been attributed to the induction of a repair mechanism by the low dose exposure. Several experiments have now been carried out on this adaptive response to better characterize the phenomenon. (A) Experiments with differential display of mRNAs indicate that human lymphocytes exposed to 2 cGy of X-rays have somewhat different mRNAs expressed than do unexposed cells. This is providing access to DNA that might be involved in adaptation. (B) Other experiments with embryonic cells from transgenic mice that are deficient in superoxide dismutase (SOD) have shown that the adaptive response is unrelated to the amount of SOD in the cells, and thus is independent of superoxide radicals. (C) Experiments in which very low doses of various restriction enzymes were electroporated into human lymphocytes have shown that low levels of double-strand DNA breaks alone are able to induce the adaptive response. (D) Experiments in which human male lymphocytes (XY chromosome constitution) and human female lymphocytes (XX chromosome constitution) were cocultivated have shown that adaptation is not caused by a change in the rate of cell progression to mitosis after a challenge dose, and is a further indication that cell stage sensitivity is not a factor in the adaptive response.

Two types of X-ray-induced radioresistance in mice: Presence of 4 dose ranges with distinct biological effects

Abstract: Preirradiation with 0.05 Gy of X rays 2 months before a second exposure to a mid-lethal dose significantly enhanced the survival rate in both female and male ICR strain mice. The radioresistance was observed between 2–2.5 months after exposure to 0.05 Gy. It did not appear within 1.5 months, and disappeared after 3 months. This radioresistance was induced only by whole-body preirradiation (not by partial irradiation of the head or the trunk). On the other hand, preirradiation with 0.30 Gy as well as 0.50 Gy resulted in radioresistance 2 weeks later, but not 2 months later. The radioresistance was induced by whole-body preirradiation or partial preirradiation of the trunk. No radioresistance was evident after exposure of intermediate preirradiation doses of 0.15 and 0.20 Gy administered before 2 months and 2–5 weeks, respectively. The present and previous results show that the biological effects of ionizing radiation may be distinguished with the following four radiation dose ranges; (1) below 0.025 Gy: no radioresistance after 2 months; (2) 0.05–0.10 Gy: significant radioresistance after 2–2.5 months; (3) 0.20 Gy: no radioresistance after 2–5 weeks; and (4) 0.30–0.50 Gy or more: significant radioresistance after 2 weeks. These results conflict with previous findings of the biological effects of ionizing radiation in which the radiation hazard increases in relation to increasing accumulated doses. Some stimulation, in addition to adaptation, by low dose irradiation may have occurred.

Mutagenic damage to mammalian cells by therapeutic alkylating agents

Abstract: Cytotoxic alkylating agents used as therapeutics include nitrogen mustards, ethyleneimines, alkyl sulfonates, nitrosoureas and triazenes. Their reactivity with DNA, RNA and proteins can cause cell death. Side-effects of treatment include tissue toxicity and secondary malignancies, likely due to the genetic damage induced. The full mutagenic potential of alkylating agents may only be realised after they undergo metabolic activation, principally by cytochromes P450. Mutagenicity is related to the ability of alkylating agents to form crosslinks and/or transfer an alkyl group to form monoadducts in DNA. The most frequent location of adducts in the DNA is at guanines. Expressed mutations involve different base substitutions, including all types of transitions and transversions. The mutational spectra of alkylating agents on mammalian cells is distinct from that induced in bacterial cells, reflecting the different codon usage by bacteria and differences in DNA repair and replication enzymes. Mutations are induced by busulfan, chlorambucil (CAB), cyclophosphamide (CP, or its metabolite), dacarbazine, mechlorethamine, melphalan, mitomycin-C (MMC), nitrosoureas and thiotepa. Although dose-dependent, the relationship is not always linear. The molarities at which alkylating agents induce cell killing and mutations vary over three orders of magnitude. The mutagenic efficiency, of alkylating agents also varies, with some agents inducing three times more mutations for equivalent cell killing. The induction of micronuclei, sister chromatid exchanges, or chromosome aberrations is variable, but has been observed for CP, CAB, MMC, melphalan and triethylenemelamine. There is insufficient information to determine whether any synergistic effects of alkylating agents used in combination will influence the cytotoxic and mutagenic damage equally. Understanding the potential synergy of alkylating agents at the cellular and molecular level should allow improvement of the therapeutic efficacy of alkylating agents without increasing the unwanted mutation induction.

Induction of chromosomal aberrations in cultured human fibroblasts by inorganic and organic arsenic compounds and the different roles of glutathione in such induction.

Abstract: Clastogenic effects of a variety of arsenic compounds were examined on cultured human fibroblasts. The following compounds were tested: inorganic arsenicals (arsenite and arsenate), the major metabolites of inorganic arsenicals in human and experimental animals [methylarsonic acid (MAA), dimethylarsinic acid (DMAA) and trimethylarsine oxide (TMAO)], and water-soluble organoarsenic derivatives [2′,3′-dihydroxypropyl-5-deoxy-5-dimethylarsinoyl-β- d -riboside (arsenosugar), arsenocholine, arsenobetaine and tetramethylarsonium iodide] found in marine organisms. Arsenic compounds induced mainly chromatid gaps and chromatid breaks. The rank order of compounds in terms of clastogenic potency was arsenite arsenate DMAA MAA TMAO. DMAA was very potent and caused chromosome pulverizations in most metaphases when present at doses higher than 7 × 10−3 M. Arsenosugar, arsenocholine, arsenobetaine and tetrametylarsonium iodide were less effective. Depletion of cellular glutathione (GSH) with l -buthionine-SR-sulfoximine (BSO), increased the incidence of chromosomal aberrations induced by arsenite, arsenate and MAA, and markedly suppressed the clastogenic effects of DMAA. DMAA was highly clastogenic even in GSH-depleted cells when the cells were incubated with DMAA in the presence of GSH (5 and 10 mM). These results suggest that GSH might play a role in protecting cells against the clastogenic effects of arsenite, arsenate and MAA. GSH might be involved in the expression of clastogenic actions of DMAA.

Mutagenicity of anticancer drugs in mammalian germ cells.

Abstract: The evidence for mammalian germ cell mutagenicity induced by anticancer drugs is summarized. Primary attention is paid to the three major mouse germ cell mutagenicity tests- the dominant lethal, heritable translocation, and morphological specific locus tests- from which most germ cell mutagenicity data historically have been obtained. Of the 21 anticancer drugs reviewed, 16 have been tested in one or more of these three tests; with all 16 tested in the most common germ cell test, the male dominant lethal test, and 9 of the 16 also tested in the female dominant lethal test. The patterns of germ cell stage specificity for most of the anticancer drugs are similar, and generally resemble the patterns seen with other types of chemicals; however, some of the patterns are unique. For example, 2 of the 8 chemicals shown to induce dominant lethal mutations in female oocytes, do not induce dominant lethal mutations in male germ cells (adriamycin and platinol). Ten of the 16 chemicals tested in the dominant lethal test were positive in post-meiotic stages (spermatids through mature sperm), and seven also induced reciprocal translocations and/or specific locus mutations in post-meiotic stages. This propensity to induce mutations in post-meiotic stages has been observed with most mutagens. However, 5 of the anticancer drugs also induced dominant lethal mutations in spermatocytes (meiotic prophase cells) and one of them, 6-mercaptopurine, uniquely induced dominant lethal mutations exclusively in preleptotene spermatocytes. Finally, three of the anticancer drugs (melphalan, mitomycin C, procarbazine) are members of a very select group of chemicals shown to induce specific locus mutations in spermatogonial stem cells of mice. The implications for human risk are discussed.

Monitoring of occupational exposure to cytostatic anticancer agents

Abstract: Many anticancer agents have been shown to be carcinogenic, mutagenic and teratogenic in experimental animals and in in vitro test systems. Epidemiological data on the association of second neoplasms with a specific chemotherapy treatment is available on some 30 agents, and in the case of 10 compounds the overall evidence on human carcinogenicity has been evaluated to be conclusive (Group 1: IARC, 1987 and 1990). The primary source of human exposure to anticancer drugs is from their use in therapy of cancer. However, persons employed in the manufacture, preparation and administration of the drugs to patients and in nursing patients may also be exposed. Safe handling of anticancer drugs, since the introduction of various general handling guidelines, is now good practice in hospitals, pharmacies and drug manufacturing companies of most developed countries. Careless handling of cancer chemotherapeutic agents may lead to exposure of the personnel in amounts detectable with chemical or biological methods in the body fluids or cell samples of the subjects. The exposure is typically to mixed compounds over long-term and to low exposure levels with accidental peaks. Therefore, the use of biological exposure markers is appropriate for the monitoring of such exposure patterns. The biological markers/methods for exposure assessment are either non-specific (e.g., cytogenetic damage, point mutations or 32P-post-labelling adducts in peripheral blood lymphocytes, urinary mutagenicity) or specific for a given compound (immunological methods for DNA adducts, specific analytical methods). Studies have revealed minor amounts of cyclophosphamide in the urine of pharmacy technicians and nurses handling the drug even when taking special safety precautions (Sessink et al. (1994a) J. Occup. Med., 36, 79; Sessink et al. (1994b) Arch. Env. Health, 49, 165). Another study showed surface wipe samples with measurable cyclophosphamide even away from the handling site (McDevitt et al. (1993) J. Occup. Med., 5, 57). These studies strongly implicate the importance of skin absorption as an exposure route. Also accidental spillage is never completely avoidable (Sorsa et al. (1988) Mutation Res., 204, 465-479). The potential confounders (smoking etc.), toxicokinetics of the agent(s) to be assessed and individual working practices should be carefully considered in any exposure assessment studies using human body fluid samples. Environmental monitoring on indicator cytostatics should be combined into studies designed to identify potential occupational exposure situations to anticancer agents. A properly performed study should also include dissemination of information to the workers to create a psychologically positive atmosphere for this important work.

Series: current issues in mutagenesis and carcinogenesis, No. 65. The genotoxicity and carcinogenicity of paracetamol: a regulatory (re)view.

Abstract: The publication of several studies reporting genotoxic effects of paracetamol, one of the world's most popular over-the-counter drugs, has raised the question of regulatory action. Paracetamol does not cause gene mutations, either in bacteria or in mammalian cells. There are, however, published data giving clear evidence that paracetamol causes chromosomal damage in vitro in mammalian cells at high concentrations and indicating that similar effects occur in vivo at high dosages. Available data point to three possible mechanisms of paracetamol-induced genotoxicity: (1) inhibition of ribonucleotide reductase; (2) increase in cytosolic and intranuclear Ca2+ levels; (3) DNA damage caused by NAPQI after glutathione depletion. All mechanisms involve dose thresholds. Studies of the relationship between genotoxicity and toxic effects in the rat (induction of micronuclei in rat bone marrow including dose-response relationship, biotransformation of paracetamol at different dosages, concomitant toxicity and biochemical markers) have recently been completed. These studies, which employed doses ranging from the dose resulting in human therapeutic peak plasma levels to highly toxic doses, give convincing evidence that genotoxic effects of paracetamol appear only at dosages inducing pronounced liver and bone marrow toxicity and that the threshold level for genotoxicity is not reached at therapeutic dosage. Reliable studies on the ability of paracetamol to affect germ cell DNA are not available. However, based on the amount of drug likely to reach germ cells and the evidence of thresholds, paracetamol is not expected to cause heritable damage in man. Various old and poorly designed long-term studies of paracetamol in the mouse and rat have given equivocal results. A few of these studies showed increased incidence of liver and bladder tumours at hepatotoxic doses. National Toxicology Program (U.S.A.) feeding studies have shown that paracetamol is non-carcinogenic when given at non-hepatotoxic doses up to 300 mg/kg/d to the rat and up to 1000 mg/kg/d to the mouse. Taking into account the knowledge of the hepatotoxicity and metabolism of paracetamol and the existence of thresholds for its genotoxicity, the animal studies do not indicate a carcinogenic potential at non-hepatotoxic dose levels. Based on this updated assessment of the genotoxicity and carcinogenicity of paracetamol, it is concluded that there is no need for regulatory action.

Effects of β- and γ-carboline derivatives on DNA topoisomerase activities

Abstract: β-Carbolines, harman (1-methyl-9 H -pyrido[3,4- b ]indole) and norharman (9 H -pyrido[3,4- b ]indole) and γ-carbolines, 3-amino-1,4-dimethyl-5 H -pyrido[4,3- b ]indole (Trp-P-1) and 3-amino-4-methyl-5 H -pyrido[4,3- b ]indole (Trp-P-2), are present in cooked foods and cigarette smoke. We studied the effects of these heterocyclic amines on the activity of DNA topoisomerases. Trp-P-1 and Trp-P-2 inhibited topoisomerase I (topo I) activity with ED 50 values of 1.48 and 1.55 μg/ml, respectively, in a relaxation assay. Harman and norharman inhibited topo I activity but with much higher ED 50 values, 23.8 and 34.4 μg/ml, respectively. Trp-P-1 and Trp-P-2 also inhibited topoisomerase II (topo II) activity at about 50 μg/ml, in a decatenation assay. Harman and norharman showed a much lower inhibitory effect on topo II activity. None of these compounds stabilized the cleavable complex mediated by topo II. Trp-P-1 and Trp-P-2 intercalated into DNA at concentrations inhibitory to topoisomerases. We considered that the intercalation with DNA and the inhibition of DNA topoisomerases by heterocyclic amines might be partly related to their inhibition of DNA excision repair and their enhancing effect on UV- or chemically induced mutagenic activity.

Mutagenic and carcinogenic properties of platinum-based anticancer drugs

Abstract: Cisplatin (DDP) is currently one of the most effective drugs for the treatment of cancer. It causes primarily intrastrand DNA-DNA cross-links, and is highly mutagenic and carcinogenic in both in vitro and in vivo experimental models. There is, however, considerable variability between the response seen in different cellular systems, probably at least partly because of the different cellular DNA repair capacities. A number of analogues of cisplatin have been developed and one of these, carboplatin (CDDCA), is also in widespread clinical use. Although it is somewhat less toxic, there is no evidence that its mode of action differs from that of cisplatin. A limited amount of mutagenicity data suggests that it has similar mutagenic and carcinogenic consequences as the parent drug. Many further analogues of cisplatin are now in clinical trials, and some of these appear to have different DNA repair responses (and therefore possibly the development of clinical resistance). Although some (e.g., iproplatin and spiroplatin) are less mutagenic than either cisplatin or carboplatin, these appear to be the ones least likely to achieve wide use. There are insufficient data on several of the most promising clinical analogues (e.g., DWA2114R and ACDDP) to judge their relative mutagenic and carcinogenic potential. Detailed studies on the DNA repair and mutagenicity characteristics of these compounds will not only provide clinically relevant data, but may also aid in the selection of further useful antitumour agents in this series.

Natural antimutagenic agents.

Abstract: Following a brief review of recent discoveries in the field of natural antimutagenic and tumor chemopreventive agents, contemporary findings in the author's laboratories employing the direct acting mutagen, ethyl methanesulfonate, in modified Ames tests and eukaryotic murine FM3A mammary tumor cells modified to be subject to thymidine-less death are described to illustrate the underlying principles. The EMS studies are illustrated with the isolation of the novel antimutagen, plicatin B, from the medicinal plants, Psoralea juncaea and P. plicata. The FM3A studies are carried out with extracts of Styrax asiatica, a plant previously studied extensively with the EMS system. The FM3A findings closely parallel the earlier work with EMS showing that the responsible agents, cinnamic acid, cinnamoyl ricinoleate and cinnamoyl cinnamate are effective both in prokaryotic and eukaryotic tests and that the new FM3A assay system has useful properties for screening and assay of novel antimutagenic agents.

Radioadaptation for gene mutation and the possible molecular mechanisms of the adaptive response.

Abstract: This paper reviews the experimental results showing that a prior exposure to a low dose of ionsing radiation induces an adaptive response expressed as a reduction of gene mutation in various cell systems. The data show that the mutagenic adaptation shares common features with the clastogenic adaptation, i.e., priming dose level, kinds of conditioning agents, time interval between conditioning and challenging treatments, degree of induced protective effect (40–75%), transitory response and inhibition by 3-aminobenzamide, a DNA repair inhibitor. Moreover, the deletion-type mutations are predominantly reduced in adapted cells, suggesting that the mechanism underlying mutagenic adaptation preferentially facilitates the removal of the DNA lesions leading to deletion-type mutations. These lesions are thought to be double-strand breaks which are likely to be also involved in the production of chromosomal damage. Recent findings on the molecular processes implicated in the cellular response to rediation provide some clues for the mechanisms that could be triggered by low-dose exposure and ultimately contribute to the protective effect. These is some evidence that the protein kinase C-mediated signalling pathway is a key step for the transduction of the low-dose-induced signal. Several recent reports indicate that the low-dose triggers changes in the expression of several genes whose products, though most of them are still not identified, would be related to DNA repair and/or control of cell cycle progression.

Chemical induction of mitotic checkpoint override in mammalian cells results in aneuploidy following a transient tetraploid state.

Abstract: Populations of tetraploid cells are found in a variety of human tumours where they may act as precursors of aneuploidy and tumorigenesis. Here we demonstrate the drug induction of tetraploid cells at mitosis by interference with cell cycle checkpoints and the coordination of mitotic events. Tetraploid cells result from mitotic exit in the absence of either chromosome segregation or cytokinesis. One class of agents that induces tetraploidy causes override of cell cycle checkpoints that require metaphase chromosome alignment as a pre-condition for initiating exit from mitosis. As a result, cells exposed to such drugs progress partially through mitosis, but exit without chromosome segregation or cytokinesis. Inhibitors of microtubule assembly comprise a second class of agents that induce tetraploidy. Many cell types are incapable of maintaining indefinite mitotic arrest in the presence of microtubule inhibitors and finally exit mitosis without microtubule dependent chromosome segregation. Inhibitors of topoisomerase II represent a third class of drugs that induce tetraploidy at mitosis. By inhibiting DNA decatenation required for sister chromatid separation at the onset of anaphase such drugs block chromosome segregation. When topoisomerase II activity is inhibited, cells nonetheless reform nuclei and exit from mitosis without chromosome segregation. Finally, inhibition of cleavage furrow formation by agents such as cytochalasins represents a fourth mechanism of tetraploidization at mitosis. We find that when Chinese Hamster Ovary cells become tetraploid, regardless of which mechanism induces this state, they are genetically unstable and become aneuploid at the subsequent mitosis. In conclusion, the failure of mitotic checkpoint function can generate gross aneuploidy from which cells with an advantage for tumor growth may be selected.

The pH 6.7 Syrian hamster embryo cell transformation assay for assessing the carcinogenic potential of chemicals

Abstract: Cell transformation models have been established for studying the cellular and molecular basis of the neoplastic process. Transformation models have also been utilized extensively for studying mechanisms of chemical carcinogenesis and, to a lesser degree, screening chemicals for their carcinogenic potential. Complexities associated with the conduct of cell transformation assays have been a significant factor in discouraging broad use of this approach despite their reported good predictivity for carcinogenicity. We previously reported that many of the experimental difficulties with the Syrian hamster embryo (SHE) cell transformation assay could be reduced or eliminated by culturing these cells at pH 6.7 culture conditions compared to the historically used pH 7.1-7.3. We and others have shown that morphological transformation (MT), the earliest recognizable phenotype in the multi-step transformation process and the endpoint used in the standard assay to indicate a chemical's transforming activity, represents a pre-neoplastic stage in this model system. In the collaborative study reported here, in which approx. 50% of the chemicals were tested under code in one laboratory (Hazelton) and the other 50% evaluated by several investigators in the second laboratory (P & G), we have evaluated 56 chemicals (30 carcinogens, 18 non-carcinogens, 8 of inconclusive carcinogenic activity) in the SHE cell transformation assay conducted at pH 6.7 culture conditions with a standardized, Good Laboratory Practices-quality protocol. An overall concordance of 85% (41/48) between SHE cell transformation and rodent bioassay results was observed with assay sensitivity of 87% (26/30) and specificity of 83% (15/18), respectively. The assay exhibited a sensitivity of 78% (14/18) for Salmonella assay negative carcinogens, supporting its value for detecting non-mutagenic carcinogens. For maximum assay sensitivity, two exposure durations were required, namely a 24-h exposure and a 7-day exposure assay. Depending on the duration of chemical treatment required to induce transformation, insight into the mechanism of transformation induction may also be gained. Based on the data reported here, as well as the larger historical dataset reviewed by Isfort et al. (1996), we conclude that the SHE cell transformation assay provides an improved method for screening chemicals for carcinogenicity relative to current standard genotoxicity assays.

The clinical use of mutagenic anticancer drugs.

Abstract: Cytotoxic chemotherapy is routinely used in the treatment of cancer, and has been an important factor in increasing 5-year survival rates for some types of this disease. A range of drugs are currently available, with differing modes of action. As well as causing some direct toxic effects, most if not all of these drugs are both mutagenic and carcinogenic. Although comparative information on these properties is generally available for anticancer drugs which alkylate DNA, it has been less readily accessible for other drug classes. This special issue contains seven reviews on the mutagenic properties of the major classes of cytotoxic drugs in clinical use, as well as one on a class of drugs that is under development. Some carcinogenicity data are also summarised, where available. Additionally, there are four more general papers, including one on the use of genetic activity profiles for comparing mutagenicity of the drugs, two on germ-cell effects, and one on biomonitoring for exposure to genotoxic anticancer drugs.

Identification of a DinB/UmuC homolog in the archeon Sulfolobus solfataricus

Abstract: To date, eight closely related homologs of the Escherichia coli UmuC protein have been identified. All of these homologs appear to play critical roles in damage-inducible mutagenesis in enterobacteriaceae. Recently, a distantly related UmuC-homolog, DinB, has also been identified in E. coli. Using the polymerase chain reaction together with degenerate primers designed against conserved regions found in UmuC-like proteins, we have identified a new member of the UmuC-superfamily in the archeon Sulfolobus solfataricus. This new homolog shows high sequence similarity to DinB and a lower level of similarity to UmuC. As a consequence, we have called this new gene dbh (dinB homolog). Analysis of approximately 2.7 kb DNA encompassing the dbh region revealed several open reading frames (orfs). One, encoding a putative ribokinase, was located immediately upstream of dbh. This orf overlaps the dbh gene by 4 bp suggesting that both proteins might be coordinately expressed. Further upstream of the ribokinase-dbh locus was another orf encoding a potential ATPase homologous to two uncharacterized S. cerevisiae proteins (YD9346.02c and SC38KCXVI_20) and another E. coli DNA repair protein, RuvB. While this is the first report of a UmuC-like homolog in an archeon, we detected additional homologs using protein sequence comparisons in Gram-positive bacteria, cyanobacteria, and among potential human EST products, indicating that UmuC-related proteins comprise a ubiquitous superfamily of proteins probably involved in DNA repair and mutagenesis.

The mutagenic properties of DNA minor-groove binding ligands

Abstract: This review summarises mutagenesis-related research on the major classes of DNA minor groove binding ligands. These compounds can bind to DNA covalently or non-covalently, and span a range of DNA sequence selectivities. Many of the non-covalent binders show effects on topoisomerase enzymes in mammalian cells, with the bisbenzimidazoles being the most active. Mutagenic effects consistent with topoisomerase inhibition are observed in vitro. Many of these compounds induce aneuploidy and polyploidy, properties which may also contribute to carcinogenic processes. Similarly, uvrA trapping by some minor groove binders may alter mutagenetic processes by inhibiting efficient repair. Distamycin has been shown to enhance the mutagenicity of ethidium bromide in bacteria by an undetermined mechanism. However, the inhibitory effects of minor groove binders on human DNA repair systems have not yet been reported. Hoechst 33258 and distamycin cause chromosome decondensation in both mouse and human cells particularly at heterochromatic regions which are rich in AT content. Various minor groove binders have been shown to induce fragile sites in cultured lymphocytes from susceptible individuals, which may have a propensity to develop particular cancers. Investigation of the relationship between fragile site inducing drugs and chromosomal rearrangements in fragile site carriers has not been investigated but may yield interesting results. Some DNA alkylating minor groove binders can generate lesions extremely toxic to mammalian cells (e.g., CC-1065 and analogues), and induce a range of DNA sequence changes in vivo, both at the site of covalent bonding as well as at surrounding sequences. This may be typical of alkylating minor groove binders which have a binding site size of several base pairs, and which stabilise helical structure. Minor groove binders have effects on gene expression in vitro by inhibiting the sequence selective binding of various transcription factors to DNA. These effects may result in expression or repression of downstream genes also. This class of ligand thus offers the possibility of mutations targeted to specific genes or genomic regions. It will be interesting to determine whether such examples of targeted mutagenesis, as has already been observed with CC-1065 and adozelesin, will result in an enhanced or in a lowered capacity to promote neoplastic disease. However it should be noted that pentamidine, a minor groove binder used in the treatment of AIDS-related PCP, has thus far shown no mutagenic effects in nuclear DNA and only a weak effect in mitochondrial DNA of yeast. These results suggest that minor groove binding does not necessarily lead to mutagenesis.

Mutagenicity, carcinogenicity and teratogenicity of antimony compounds.

Abstract: The paper reviews the information available concerning the mutagenic, teratogenic and carcinogenic effects of antimony. A claim that antimony compounds could have mutagenic properties is based on insufficient and not particularly relevant data. Additional experiments, particularly with organic antimony compounds, would be desirable, but from what we know already, one may be confident that antimony is less a mutagenic risk than many other metals such as As, Cr, Ni, among others. Evidence for a carcinogenic risk of antimony in experimental animals was judged by the IARC sufficient for antimony trioxide and limited for antimony trisulfide. In man, IARC considered antimony trioxide as possibly carcinogenic. However, exposure in all studies on which these conclusions are based also involved other proven or likely carcinogenic compounds. Studies with pure antimony compounds, especially those used in therapy, need to be performed to clarify the situation. Although some indications exist that antimony trioxide could interfere with embryonic and fetal development, the studies seem not entirely conclusive. It is regrettable that, at least to our knowledge, the outcome of pregnancy in women treated with antimony compounds for leishmaniasis has not been studied. In conclusion, it appears that mutagenic, carcinogenic and teratogenic risks of antimony compounds, if they exists at all, are not very important.

The beneficial effects of dietary restriction: reduced oxidative damage and enhanced apoptosis.

Abstract: There is compelling evidence for the central role of oxidative damage in the aging process and for the participation of reactive oxygen species in tumor initiation and promotion. Caloric restriction (CR) or energy restriction retards age-associated increases in mitochondrial free-radical production and reduces the accumulation of oxidatively damaged cell components. CR has also been shown to slow down age-related declines in various repair capabilities, including some types of DNA repair. It is proposed that inhibitors of mitochondrial electron transport and/or uncouplers of oxidative phosphorylation (rotenone, amytal, amiodarone, valinomycin, etc.), when used at extremely low doses, could mimic the effects of CR in model systems. The objective is to lower mitochondrial free-radical production by decreasing the fraction of electron carriers in the reduced state. In addition to a variety of other effects, CR has been shown to increase the rate of apoptosis, particularly in preneoplastic cells, and in general, to promote elevated levels of free glucocorticoids (GCs). GCs are known to induce tissue-specific apoptosis and to upregulate gap-junction-mediated intercellular communication (GJIC). Tumor promoters like phorbol esters have the opposite effect, in that they inhibit both the process of apoptosis and GJIC. The enzyme poly (ADP-ribose) polymerase (PARP) is thought to play a central role in apoptosis, in a manner that has been highly conserved in evolution. There is good evidence that the apoptosis-associated Ca/Mg-dependent DNA endonuclease is maintained in a latent form by being poly (ADP-ribosylated). Apoptosis would require the removal of this polymer from the endonuclease, and, most likely, its removal from topoisomerase II and histone H1 as well. The role of poly (ADP-ribose) in apoptosis, carcinogenesis, and aging could be studied by the use of modulators of PARP activity (3-aminobenzamide, 3-nitrosobenzamide, 1% ethanol, etc.), inhibitors of poly ADP-ribose) glycohydrolase activity (ethacridine, 43 degrees C, etc.), and inhibitors of the PARP-specific protease (interleukin-1 beta converting enzyme (ICE)-like protease). Also, it would be of interest to determine if CR can decrease the half-life of poly (ADP-ribose), upregulate GJIC, and modulate the activities of PARP, the glycohydrolase, and the PARP-specific protease, factors potentially important in these processes.

Studies on the role of specific dietary fibres in protection against colorectal cancer

Abstract: Although dietary fibre is generally thought to protect against the development of coloreactal cancer, some of the results of animal and epidemiological studies are equivocal. We believe that this may be because the term dietary fibre covers a range of complex materials and some may protect but others may not. Dietary fibre is mainly composed of plant cell walls which vary in composition and properties according cell type and plant species. In addition to polysaccharides, the walls of some plant cell types contain the hydrophobic polymers lignin or suberin. Two groups of mechanisms have been proposed for the way dietary fibres may protect against colorectal cancer: those in which the dietary fibre may act directly and those in which the dietary fibre may have an indirect effect as a consequence of it being degraded by colonic bacterial enzymes and the products fermented. Direct mechanisms include the adsorption of carcinogens onto undergraded dietary fibres which pass out of the intestinal tract in the faeces. We have shown that different types of plant cell walls adsorbed a range of carcinogens, including heterocyclic aromatic amines, to different extents. Cell walls that contained lignin or suberin adsorbed hydrophobic carcinogens particularly well. Furthermore, the presence of lignin, and probably suberin, in the walls makes them resistant to degradation in the colon. Wheat bran, which is a good source of dietary fibre, contains some cell types with lignified walls. We used Fischer-344 rats to test the ability of wheat bran to protect against the formation of aberrant crypts (which are considered to be precursors to colon cancer) caused by the heterocyclic aromatic amine, 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ). Our results indicate that wheat bran protects and probably does so by a direct mechanism.

Activity profiles of antimutagens: in vitro and in vivo data.

Abstract: In this review, retinol, chlorophyllin, and N-acetylcysteine are examined and compared with regard to their antimutagenic activity against some promutagens and a group of direct-acting alkylating agents. The promutagens included aflatoxin B1, certain polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), and certain heterocyclic amines (e.g., food pyrolysates). Results of antimutagenicity testing selected from data surveyed in the published literature are displayed graphically as activity profiles of antimutagens showing both the doses tested and the extent of inhibition or enhancement of mutagenic activity. All three antimutagens are discussed in terms of their putative mechanisms of action in vitro and in vivo with emphasis on the xenobiotic metabolizing enzyme systems.