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Showing papers in "Mutation Research in 1993"


Journal ArticleDOI
TL;DR: The versatility and simplicity of the CBMN assay together with new developments in automation should ensure its successful application in monitoring exposed populations as well as in identifying mutagen-sensitive individuals within a population.
Abstract: The development of the cytokinesis-block (CB) technique has transformed the human-lymphocyte micronucleus assay (MN) into a reliable and precise method for assessing chromosome damage. Recent studies in our laboratory have confirmed that this method is a sensitive indicator of in vivo radiation exposure in (a) patients undergoing fractionated partial-body radiotherapy and (b) rodents exposed to uniform whole-body irradiation, thus supporting the application of the cytokinesis-block micronucleus (CBMN) assay for biological dosimetry. To further define the use of this assay in biomonitoring we performed extensive studies to determine the spontaneous level of MN in normal human populations and its relationship to various life-style factors. We have also developed a new variation to the CBMN assay that permits the conversion of excision-repairable lesions to MN within one cell-cycle using cytosine arabinoside. With this method the slope of the in vitro dose-response curves was increased by a factor of 1.8 for X-rays, 10.3 for ultraviolet (UV, 254 nm) radiation and approximately 40-fold for methylnitrosourea. Consequently the CBMN assay can now be used to measure not only whole chromosome loss or chromosome breaks but also excision-repair events. The versatility and simplicity of the CBMN assay together with new developments in automation should ensure its successful application in monitoring exposed populations as well as in identifying mutagen-sensitive individuals within a population.

953 citations


Journal ArticleDOI
TL;DR: The development of the SCGE assay, existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA, and the potential applications of the technique are reviewed.
Abstract: The single cell gel electrophoresis (SCGE) assay is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells. Here we review the development of the SCGE assay (with particular reference to the alkaline version), existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA, and the potential applications of the technique.

894 citations


Journal ArticleDOI
TL;DR: The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV and actively proliferate during about 50% of the epithelial cycle.
Abstract: In whole mounts of seminiferous tubules of C3H/101 F1 hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis. The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A1 plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the Apr and all of the Aal spermatogonia differentiate into A1 spermatogonia. It was estimated that there are 2.5 x 10(6) differentiating spermatogonia and 3.3 x 10(5) undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycle.

526 citations


Journal ArticleDOI
TL;DR: Little is known about the ability of DNA repair to preserve the normal pattern of methylation in somatic cells, but mutations in the p53 gene in tumours are also very commonly in 5mCpG doublets.
Abstract: 5-Methylcytosine (5mC) in DNA is produced by post-synthetic modification of cytosine residues, and it occurs primarily in CpG doublets in the mammalian genome. 5mC is a mutable site, because it can undergo spontaneous deamination to thymine. There is a repair mechanism which specifically recognises G.T mispairs, and replaces thymine with cytosine. However, this repair is not fully efficient, because the 5mC-->T transition mutation occurs about 10 times as frequently as other transitions. Such mutations are frequently seen in inherited diseases, and mutations in the p53 gene in tumours are also very commonly in 5mCpG doublets. As well as mutations, there can also be heritable changes in DNA methylation, known as epimutations, which may be of particular significance in somatic cells. Whereas the pattern of DNA methylation is very constant for any one cell type, the pattern becomes very variable in tumour cells. The breakdown of the normal controls of DNA methylation in tumorigenesis can lead to increased gene expression or to gene silencing. DNA damage increases not only mutation, but also heritable changes in methylation. At present, little is known about the ability of DNA repair to preserve the normal pattern of methylation in somatic cells.

333 citations


Journal ArticleDOI
TL;DR: This review covers the methods used for detection of unknown mutations, namely the ribonuclease, denaturing gradient-gel electrophoresis, carbodiimide, chemical cleavage, single-strand conformation polymorphism, heteroduplex and sequencing methods.
Abstract: Mutation detection is important in all areas of biology. Detection of unknown mutations can involve sequencing of kilobases of DNA, often in many patients. This has lead to the development of methods to screen DNA for mutations as well as methods to detect previously described mutations. This review discusses current methods used for such purposes with special emphasis on genetic diseases of humans. However, savings can be made by similar means in other areas of biology where repetitive or extensive sequencing for comparative purposes needs to be done. This review covers the methods used for detection of unknown mutations, namely the ribonuclease, denaturing gradient-gel electrophoresis, carbodiimide, chemical cleavage, single-strand conformation polymorphism, heteroduplex and sequencing methods. Once mutations have been defined they can be searched for repeatedly by methods referred to as diagnostic methods. Such methods include allele-specific oligonucleotide hybridization, allele-specific amplification, ligation, primer extension and the artificial introduction of restriction sites. We can now choose from a range of excellent methods, but the choice will usually depend on the background of the laboratory and/or the application in hand. Screening methods are evolving to more satisfactory forms, and the diagnostic methods can be automated to screen whole populations inexpensively.

313 citations


Journal ArticleDOI
TL;DR: Transgenic rats containing the same lambda/lacI shuttle vector have been developed for inter-species comparison of mutagenesis testing results, which may offer a better understanding of the specific mechanisms involved in Mutagenesis at the molecular level in vivo.
Abstract: Transgenic mice carrying shuttle vectors containing the lacI gene as the target permit the in vivo measurement of mutations in multiple tissues and have been used to test the mutagenic effects of several compounds. Tissue-specific and time-dependent responses have been observed, and the spectrum of mutations determined by sequencing allows analysis of the role of expression time in mutagenesis. The results obtained from sequencing analysis have demonstrated spectra paralleling those observed in alternative in vivo assays. In addition to color screening, modifications to this system have permitted direct selection for mutations in the lacI target by a variety of methods. Transgenic rats containing the same lambda/lacI shuttle vector have been developed for inter-species comparison of mutagenesis testing results, which may offer a better understanding of the specific mechanisms involved in mutagenesis at the molecular level in vivo.

174 citations


Journal ArticleDOI
TL;DR: The inhibition mechanisms of flavone, kaempferol, morin, flavanone, and 2'-hydroxyflavanone were concentration dependent, being competitive at low concentrations and mixed or non-competitive at concentrations about the ID50 value.
Abstract: Sixty-four flavonoids were tested for their antimutagenic potencies with respect to IQ in Salmonella typhimurium TA98 and in part also towards MeIQ, MeIQx, Trp-P-2, and Glu-P-1 and in S. typhimurium TA100. Antimutagenic potencies were quantified by the inhibitory dose for 50% reduction of mutagenic activity (ID 50 ). A carbonyl function at C-4 of the flavane nucleus seems to be essential for antimutagenicity: two flavanols and four anthocyanidines were inactive. Again, five isoflavons, except biochanin A, were inactive. Within the other groups of 21 flavones, 16 flavonols and 16 flavanones the parent compounds flavone, flavonol, and flavonone possessed the highest antimutagenic potencies (ID 50 : 4.1, 2.5, 5.5 nmoles). Increasing polarity by introduction of hydroxyl functions reduced antimutagenic potency. Reducing polarity of hydroxy flavonoids by methyl etherification, however, increased antimugenic potency again. 6-Hydroxy- and 2′-hydroxy substituted flavonoids were considerably less potent antimutagens. Of 11 flavonoid glycosides tested all compounds except apigenin- and luteolin-7-glucoside (ID 50 :74, 115 nmoles) were inactvie or only weakly antimutagenic. Rings C and A of the nucleus were not essential for antimutagenicity: chalcone and three derivatives were nearly as active as comparable flavones while antimutagenicity of benzylidenacetone was considerably redcued (ID 50 : 95 nmoles). Cinnamylaldehyde and cinnamoates, however, were inactive. A planar structure in the vacinity of the carbonyl group may also be important for antimutagenicity. Flavanones were less potent antimutagens than the corresponding flavones, but dihydrochalcones and 14 structurally related saturated aromatic carbonyl compounds were inactive. Fisetin and 6-hydroxyflavone were competitive inhibitors, but luteolin was a mixed type inhibitor. The inhibition mechanisms of flavone, kaempferol, morin, flavanone, and 2′-hydroxyflavanone were concentration dependent, being competitive at low concentrations and mixed or non-competitive (2′-hydroxyflavanone) at concentrations about the ID 50 value. No fundamental differences between the two tester strains and no clear influence of mutagen structure on antimutagenic potency could be detected.

173 citations


Journal ArticleDOI
TL;DR: The structure of the aldehydes investigated appears to influence the cyto- and genotoxic potential in the following ways: the length of the lipophilic tail has no influence on chromosomal aberration induction, but appears to determine the yield of SCE and micronuclei, and the cytot toxic potential.
Abstract: 4-Hydroxynonenal (HNE), one of the major products of lipid peroxidation, has been demonstrated to induce genotoxic effects in the micromolar range. HNE has too structural domains, a lipophilic tail and a polar head with three functional groups: the aldehyde and hydroxy groups and the trans CC double bond. To evaluate their relative importance, the genotoxic effects of HNE were compared with those of the homologous aldehydes 4-hydroxyhexenal and 4-hydroxyundecenal (different lengths of the lipophilic tail), and the analogous aldehydes 2-trans-nonenal (lacking the OH group) and nonanal (lacking the OH group and the trans CC double bond). This investigation was carried out on primary cultures of adult rat hepatocytes in order to further determine the influence of biotransformation- and/or detoxification reactions. A 3-h treatment with HNE induces statistically significant levels of SCE at concentrations > or = 0.1 microM, micronuclei at concentrations > or = 1 microM and chromosomal aberrations at a concentration of 10 microM. Compared to HNE the homologous aldehydes induced a significant genotoxic effect at higher concentrations. Statistically significant increases in SCE frequency were obtained at concentrations > or = 1 microM for 4-hydroxyundecenal and at a concentration of 10 microM for 4-hydroxyhexenal. The induction of chromosomal aberrations was significantly elevated at concentrations of > or = 10 microM and 10 microM for 4-hydroxyhexenal and 4-hydroxyundecenal, respectively. Except for a 4-hydroxyhexenal concentration of 1 microM, both aldehydes did not induce statistically significant levels of micronuclei. The HNE analogous aldehydes 2-trans-nonenal and nonanal induced statistically significant frequencies of SCE at concentrations of > or = 1 microM (nonanal) and > or = 10 microM (2-trans-nonenal). No significant induction of chromosomal aberrations or micronuclei could be demonstrated. The structure of the aldehydes investigated appears to influence the cyto- and genotoxic potential in the following ways. (1) The length of the lipophilic tail has no influence on chromosomal aberration induction, but appears to determine the yield of SCE and micronuclei, and the cytotoxic potential. (2) The lack of the OH group (2-trans-nonenal) reduces the SCE-inducing potential of the aldehyde shifting the dose-effect curve to higher concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

171 citations


Journal ArticleDOI
TL;DR: It is concluded that putative genotoxic rodent carcinogenesis can be correlated both with chemical structure and the extent and nature of the induced effect, and that it is of clear relevance to humans.
Abstract: Gold and her colleagues have tabulated the results of rodent bioassays on 552 chemicals and have analysed the data. The present study complements those analyses by providing a perspective from the viewpoint of the chemical structure of the carcinogens. The chemical structure of each of the carcinogens is displayed and the Gold database is represented with the test agents as the primary variable. The carcinogens are gathered into six chemical classes and each chemical is assessedfor structural alerts to DNA reactivity. The database is then analysed using an integration of the following parameters: bioassay in rat, mouse or both; structural alert status; chemical class; sites and multiplicity of carcinogenesis, and trans-species carcinogenicity. A series of Figures is presented that enables rapid acquaintance with what represent the core database of rodent carcinogenicity. The several analyses presented combine in endorsing the reality of two broad classes of rodent carcinogen — presumed DNA-reactive and others (putative genotoxic and non-genotoxic carcinogens, but semantics have been largely avoided). Vainio and his colleagues have tabulated 55 situations in which humans have succumbed to chemically induced cancer, and have listed the tissues affected. This database of human carcinogens has been anaylysed in the present study as done for the rodent carcinogen database, and comparisons made between the two. The predominance of putative genotoxic carcinogens in the human database was confirmed, as was the reality of putative non-genotoxic carcinogenicity in humans. It is concluded that putative genotoxic rodent carcinogenesis can be correlated both with chemical structure and the extent and nature of the induced effect, and that it is of clear relevance to humans. In contrast, it is concluded that putative non-genotoxic rodent carcinogenesis is more closely related to the test species than to the test chemical, and that it is essentially unpredictable in the absence of mechanistic models. In the absence of such models non-genotoxic Carcinogenic effects should be extrapolated to humans with caution. Progress in the accurate prediction and extrapolation of rodent carcinogenicity will be helped by a common, if only temporary, enabling acceptance that not all carcinogens are intrinsically genotoxic.

167 citations


Journal ArticleDOI
TL;DR: Dietary fibre has a complex and highly variable composition, and although some dietary fibres may protect against colorectal cancer, it is unlikely that all are equally protective.
Abstract: Dietary fibre has a complex and highly variable composition. Although some dietary fibres may protect against colorectal cancer, it is unlikely that all are equally protective. Dietary fibre is principally composed of plant cell walls, but it also includes components obtained from cell walls (e.g. cellulose, pectin, and lignin), and non-starch polysaccharides (NSPs) from other sources (e.g. seaweeds and micro-organisms). The AOAC and Englyst methods are commonly used to determine the total amount of dietary fibre in foods. Most of the cell walls in food plants are from parenchyma cells, which are extensively degraded by bacteria in the colon. Cell types with walls containing the hydrophobic polymers lignin, suberin, or cutin also occur in food plants in small numbers, but they may be important in preventing colorectal cancer. Lignin, and possibly the other polymers, protect these walls from degradation. Epidemiological, human intervention, and animal studies can be used to try to identify the most protective dietary fibres. Epidemiological studies are difficult to interpret because usually only the total amount of dietary fibre eaten is reported. Intervention studies indicate that wheat bran dietary fibre may be protective. The results of animal carcinogenesis studies are variable, but sources of insoluble dietary fibres, including wheat bran, appear more protective than soluble dietary fibres, and some dietary fibres appear to enhance carcinogenesis. Possible mechanisms for protection by dietary fibres can be divided into two groups: those where the dietary fibre is acting directly, and those which result from the dietary fibre being degraded by colonic bacterial enzymes and the products fermented. Possible direct mechanisms include the binding of carcinogens to undegradable dietary fibres, and the absorption of water by undegradable dietary fibre resulting in increased faecal bulk and shortened transit times. Possible indirect mechanisms include the lowering of the colon pH by the short-chain fatty acids produced by bacterial fermentation, and the specific effects of butyrate. There are also a number of possible mechanisms by which some dietary fibres may enhance carcinogenesis. Use of better defined dietary fibres will increase our understanding of the role of dietary fibres in modulating colorectal cancer.

167 citations


Journal ArticleDOI
TL;DR: Application of the Ames test permitted the delimitation of three areas of influence of the petrochemical industrial complex, and the test proved to be adequate for the detection of contaminants from thepetrochemical industry.
Abstract: The present study was carried out on the waters of the Cai River (Rio Grande do Sul, Brazil) in an area under the influence of a petrochemical industrial complex, as the continuation of a study in which the mutagenic activity of water samples was evaluated in the internal area of this complex. In the previous study, the release of inducing substances was detected, revealing the need for a full analysis of the real ecological impact of the industrial complex on the river. Water samples from different sites along the Cai River were subjected to the Ames test during a study of 20 months duration for the detection of possible mutagens. Strains TA100 and TA98 were used for initial sample screening in the presence and absence of the S9 mix at a standard dose of 2000 μl/plate. When positive activity (values equal to twice the spontaneous mutation rate) and/or cytotoxic activity (cell survival below 60%) was detected, the dose-response relationship was studied. Thirty-four percent of the samples tested were mutagenic, with different values according to collection site. Of the total number of positive responses, 6% were obtained for samples collected at the blank site upstream from the area studied, 82% at sites closest to the industrial complex, and 12% in downstream areas. Strain TA98 was the most sensitive in assays with no metabolic activation. A low frequency of induction (2%) was observed for strain TA102. Application of the Ames test permitted the delimitation of three areas of influence of the petrochemical industrial complex, and the test proved to be adequate for the detection of contaminants from the petrochemical industry.

Journal ArticleDOI
TL;DR: The finding that the adducts are not quantitatively and qualitatively the same in the three organs examined is likely due to differences of metabolism in these organs, leading to different ultimate carcinogens and may also result from differences in the efficiency of repair processes.
Abstract: Ochratoxin A (OTA) is a mycotoxin which has been implicated in Balkan endemic nephropathy, a disease characterized by a high incidence of urinary tract tumors. It induces DNA single-strand breaks and has been shown to be carcinogenic in two rodent species. For a better understanding of the OTA genotoxic effect, OTA-DNA adduct formation and disappearance has been measured using the 32P-post-labelling method after oral administration of 2.5 mg/kg of OTA to mice. In kidney, liver and spleen, several modified nucleotides were clearly detected in DNA, 24 h after administration of OTA, but their level varied significantly in a tissue and time dependent manner over a 16-day period. Total DNA adducts reached a maximum at 48 h when 103, 42 and 2.2 adducts per 109 nucleotides were found respectively in kidney, liver and spleen, indicating that kidney is the main target of the genotoxicity and likely carcinogenicity of OTA. The major adduct differed between kidney and liver. All adducts disappeared in liver and spleen 5 days after compound administration, whereas some adducts persisted for at least 16 days in the kidney. Some adducts were organ specific. The finding that the adducts are not quantitatively and qualitatively the same in the three organs examined is likely due to differences of metabolism in these organs, leading to different ultimate carcinogens and may also result from differences in the efficiency of repair processes.

Journal ArticleDOI
TL;DR: It is demonstrated that a surprisingly high proportion of T-cells with stable and often complex irradiation-induced chromosome aberrations are able to proliferate and form expanding cell clones in vitro and that X-irradiation induces latent chromosome damage and genomic instability in human T-lymphocytes.
Abstract: To study the effects of X-irradiation on clone forming ability and karyotypic abnormalities in human peripheral blood lymphocytes, cells were exposed to 3 Gy of X-rays in vitro and either individual T-cell clones or long-term T-cell cultures were established. The karyotypes were analyzed in G-banded chromosome preparations after proliferation for 9–34 days in vitro. T-cell clonal karyotype abnormalities were found in 24 of 37 (65%) irradiated and in two of 43 (5%) control clones. Balanced reciprocal translocations and deletions were the predominating types of clonal aberrations. Complex aberrations and unstable karyotypes were found in about half of the irradiated clones. Some of the T-cell clones demonstrated sequential change from normal to aberrant karyotype. Other clones seemed to develop multiple, heterogeneous chromosomal aberrations during growth in vitro. X-Irradiated T-cells grown in long-term T-cell culture displayed karyotype abnormalities in 60–80% of the cells, and the types of aberrations were similar to those found in the individual, irradiated T-cell clones. An increasing number of cells with the same abnormal karyotype was observed when the cultivation time was extended, indicating clonal proliferation. These results demonstrate that a surprisingly high proportion of T-cells with stable and often complex irradiation-induced chromosome aberrations are able to proliferate and form expanding cell clones in vitro. Furthermore, the results indicate that X-irradiation induces latent chromosome damage and genomic instability in human T-lymphocytes.

Journal ArticleDOI
TL;DR: PCR Amplification of Specific Alleles shows promise for population screening because the technique is rapid, highly reproducible, inexpensive, nonisotopic, and amendable to automation.
Abstract: We review a method termed PCR Amplification of Specific Alleles (PASA), a generally applicable technique for the detection of known point mutations, small deletions and insertions, polymorphisms and other sequence variations. PASA is a modification of PCR that depends on the synthesis of a PCR oligonucleotide primer that precisely matches with one of the alleles but mismatches with the other. When the mismatch occurs near the 3' end of the PCR primer, amplification is inefficient. Therefore, preferential amplification of the perfectly matched allele is obtained. The method should be generally applicable as our results indicate that with proper optimization all possible alleles can be reliably distinguished. The ease and technical simplicity of PASA make genetic analyses more accessible. PASA can be also adapted to accommodate specific requirements and can be extended by incorporating other techniques. Moreover, PASA shows promise for population screening because the technique is rapid, highly reproducible, inexpensive, nonisotopic, and amendable to automation.

Journal ArticleDOI
TL;DR: It is established that PhIP causes increased cell proliferation in colon mucosa but not in the non-target liver or kidney of male rats, and exposure to HCAs should accordingly be avoided as far as possible.
Abstract: The diet contains various mutagens and carcinogens that can be classified into three groups: naturally occurring chemicals, synthetic compounds and compounds produced by cooking. The first group includes mycotoxins and plant alkaloids while the second is exemplified by food additives and pesticides. The third includes polycyclic aromatic hydrocarbons and heterocyclic amines (HCAs). HCAs are mutagenic to microbes and eukaryotes and their precursors are creatine or creatinine, sugars, and amino acids in meat and fish. Among 10 HCAs so far examined for carcinogenicity in rodents, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced colon cancer in rats. PhIP is an especially interesting compound inducing colon tumors specifically in male F344 rats and only very rarely in females, which develop mammary carcinomas at high frequency instead. Since induced DNA adduct levels, determined by the 32P-postlabeling method, were found to be almost the same in male and female F344 rats adduct formation in itself is not directly responsible for carcinogenesis. We established, however, that PhIP causes increased cell proliferation in colon mucosa but not in the non-target liver or kidney of male rats. Induction of cell proliferation is therefore possibly an additional important factor determining carcinogenic organ specificity. In terms of molecular alteration ras family gene mutations are very rare and no mutations are evident in the p53 gene in colon tumors induced by HCAs. Their development due to HCAs can thus be considered an appropriate experimental model for human colon tumors in which ras or p53 gene activation is not involved. Since HCAs are genotoxic compounds, a causal role in some stage of human colon carcinogenesis is plausible. Exposure to HCAs should accordingly be avoided as far as possible.

Journal ArticleDOI
TL;DR: A sex-related effect in the induction of micronuclei is suggested and a dose-response relationship with duration of exposure was apparent with a maximum increment of 71% in the frequency of micronsuclei in subjects exposed for over 30 years.
Abstract: A great deal of the flower cultivation in Italy is carried out in the western part of the Region of Liguria. The extensive use of pesticides professionally exposes floriculturists operating in this area to a complex mixture of compounds. The frequency of micronuclei in peripheral lymphocytes has been evaluated in 71 floriculturists and in a control group of 75 healthy blood donors living in the area. No correlation between age and micronucleus frequency was found in peripheral lymphocytes of the controls while an increase in this parameter was observed in the elderly of the exposed group. Our data suggest a sex-related effect in the induction of micronuclei. The frequencies of micronucleated lymphocytes were significantly higher in females than in males in both exposed and control groups (RR = 1.45, 95% C.I. 1.25-1.67). The main result of this study, however, is the observation of a significant increase in micronucleated lymphocyte frequency in people occupationally exposed to pesticides. The micronucleus frequency was 8.57/1000 for exposed persons and 6.67/1000 for controls (p < 0.05). A dose-response relationship with duration of exposure was apparent with a maximum increment of 71% in the frequency of micronuclei in subjects exposed for over 30 years.

Journal ArticleDOI
TL;DR: A comparison of target organs for mutagens and non-mutagens is presented for 351 rodent carcinogens in the Carcinogenic Potency Database (CPDB) with mutagenicity evaluations in Salmonella, showing results consistent with the hypotheses that in high-dose rodent tests mitogenesis is important in the carcinogenic response for mutAGens andNon-Mutagens alike.
Abstract: A comparison of target organs for mutagens and non-mutagens is presented for 351 rodent carcinogens in the Carcinogenic Potency Database (CPDB) with mutagenicity evaluations in Salmonella. Results are consistent with the hypotheses that in high-dose rodent tests mitogenesis is important in the carcinogenic response for mutagens and non-mutagens alike, and that mutagens have a multiplicative interaction for carcinogenicity because they can both damage DNA directly and cause cell division at high-doses. These hypotheses would lead one to expect several results that are found in the analysis: First , a high proportion of both mutagens and non-mutagens induce tumors in rodent bioassays at the MTD. Second , mutagens compared to non-mutagens are: (a) more likely to be carcinogenic; (b) more likely to induce tumors at multiple target sites; and (c) more likely to be carcinogenic in two species. Among carcinogens that induce tumors at multiple sites in both rats and mice, 81% are mutagens; in comparison, among carcinogens that are positive at only a single target site in one species and are negative in the other, 42% are mutagens. Since tissue distribution and pharmacokinetics would not be expected to differ systematically between mutagens and non-mutagens, one would not expect systematic differences in the particular organs in which tumors are induced. Results do not support the idea that mutagens and non-mutagens induce tumors in different target organs. Both mutagens and non-mutagens induce tumors in a wide variety of sites, and most organs are target sites for both. Moreover, the same tend to be the most common sites for both: 79% or more both mutagenic and non-mutagenic carcinogens are positive in each species in at least one of the 8 most frequent target sites: liver, lung mammary gland, stomach, vascular system, kidney, hematopoietic system and urinary bladder. Species differences are discussed as well as results for particular target organs: liver, Zymbal's gland and kidney.

Journal ArticleDOI
TL;DR: It is concluded that trapping by complex formation plays a role in the antimutagenic actions of chlorophyllin against many mutagens, particularly notable being the actions against ICR-170, quinacrine, aflatoxin B1, Trp-P-1 and Trp -P-2.
Abstract: Chlorophyllin is known to inhibit the mutagenicity of a variety of compounds. Using highly purified samples of chlorophyllin and its family compounds, we studied the mechanism of the inhibition. Since mutagens with polycyclic planar structures are particularly strongly inhibited, it seemed likely that the inhibition arises by trapping of the mutagens by chlorophyllin through complex formation at the planar surfaces of these molecules. To explore this possibility, we prepared a Sepharose bearing covalently linked chlorophyllin as ligand, and the adsorption of mutagens to this Sepharose was measured. Three different chlorophyllin derivatives were used, i.e., copper-chlorin, iron-chlorin and chlorin, to investigate the role of metal in the center of the chlorophyllin chromophore. Adsorption of 37 different compounds, mostly mutagens, in 0.02 M Tris-HCl buffer at pH 8.0 to these chlorophyllin-Sepharose preparations was studied in a quantitative manner. The results showed that most of the compounds having three or more fused rings were strongly adsorbed with apparent dissociation constants of 10(-5)-10(-6) M, whereas those having two fused rings or one ring were only poorly adsorbed. Since the three Sepharose adsorbents gave similar adsorption profiles, it appeared that the central metal in the chlorophyllin molecule does not play a crucial role in the adsorption. We also measured the inhibitory effect of copper-chlorin against the mutagenicity of some of these compounds using the Salmonella assay. The results showed that those mutagens that were strongly adsorbable to copper-chlorin-Sepharose were subject to efficient inhibition by copper-chlorin, whereas many of those only poorly adsorbed were inhibited only weakly. We concluded that trapping by complex formation plays a role in the antimutagenic actions of chlorophyllin against many mutagens, particularly notable being the actions against ICR-170, quinacrine, aflatoxin B1, Trp-P-1 and Trp-P-2. An unusual behavior of Trp-P-2 in the adsorption process, i.e., a very tight complex formation at an extremely low Trp-P-2 concentration, was found; the implication of this phenomenon in relation to the real environmental setting is discussed.

Journal ArticleDOI
J. Rank1, A.-G. Jensen1, B. Skov1, L.H. Pedersen1, K. Jensen1 
TL;DR: The genotoxic potential of the herbicide Roundup and its active agent, glyphosate isopropylamine salt, was studied in three different assays and a significant increase in chromosome aberrations appeared after treatment with Roundup.
Abstract: The genotoxic potential of the herbicide Roundup and its active agent, glyphosate isopropylamine salt, was studied in three different assays. No clastogenic effects were found in the mouse bone marrow micronucleus test for either of the two agents. In the Salmonella assay only Roundup was tested. It showed a weak mutagenic effect for the concentrations 360 micrograms/plate in TA98 (without S9) and 720 micrograms/plate in TA100 (with S9). These concentrations are close to the toxic level. The anaphase-telophase Allium test showed no effect for the glyphosate isopropylamine salt, but a significant increase in chromosome aberrations appeared after treatment with Roundup at concentrations of 1.44 and 2.88 mg/l when calculated as glyphosate isopropylamine. The most frequent aberrations observed could be characterized as disturbances of the spindle.

Journal ArticleDOI
TL;DR: The aberrant crypt focus (ACF) assay is used to test and develop hypotheses linking diet and colon cancer, which include a risk associated with 5-hydroxymethyl-2-furaldehyde in caramelized sugar, a Risk associated with some factor in thermolyzed casein, and a single nutrient boluses of sucrose and fructose.
Abstract: We have used the aberrant crypt focus (ACF) assay to test and develop hypotheses linking diet and colon cancer. The hypotheses were suggested by epidemiological studies that identified possible dietary factors associated with colorectal cancer risk. The ACF assay was used to quantitate the effect of the dietary factors on the initiation and growth of these putative precursors of colon cancers in experimental animals. Using this approach we have developed 3 new hypotheses for the role of diet in colorectal cancer. These are (1) a risk associated with 5-hydroxymethyl-2-furaldehyde in caramelized sugar, (2) a risk associated with some factor in thermolyzed casein, and (3) a risk associated with single nutrient boluses of sucrose and fructose. The importance of these hypotheses has still to be tested in long term carcinogenesis experiments, in analytic epidemiology studies and then, perhaps, in intervention trials.

Journal ArticleDOI
TL;DR: A range of test chemicals were selected and used to evaluate the ability of test systems ranging from tubulin polymerisation, fungal cultures, cultured mammalian cells and intact rodents to detect chemical aneugens and to assess the significance of such activity to exposed human populations.
Abstract: Within the framework of its' Environment Research and Development Programme, the European Communities (EC) Directorate General (DG) XII has supported a research project aimed at developing and validating assay systems for the detection and evaluation of chemicals capable of inducing numerical chromosome changes such as aneuploidy and polyploidy. A range of test chemicals were selected, which include a core set comprising; colchicine, econazole nitrate, chloral hydrate, hydroquinone, diazepam, thiabendazole, cadmium chloride, thimerosol, pyrimethamine and vinblastine sulphate. These test chemicals were used to evaluate the ability of test systems ranging from tubulin polymerisation, fungal cultures, cultured mammalian cells and intact rodents to detect chemical aneugens and to assess the significance of such activity to exposed human populations.

Journal ArticleDOI
TL;DR: It is concluded that most chromosome lesions directly induced by heavy ions are hardly compatible with cell survival and thus disappear after a few cell generations, however, surviving cells acquire a de novo chromosome instability leading to the formation of clones with unbalanced karyotypes at late passages.
Abstract: Cultures of human skin fibroblasts were exposed to heavy ions: neon (E = 10.74 MeV/u) and argon (E = 10.52 MeV/u) at fluences of 10(6), 2 x 10(6) and 4 x 10(6) and lead (E = 9.5 MeV/u) at a fluence of 2 x 10(6) particles/cm2. Cultures were further prolonged for up to 25 passages and karyotyping was performed at various times. Radiation-induced chromosome anomalies progressively decreased, became quite rare at passages 5-7 and increased at later passages. Around passages 20-25, most anomalies occurring were dicentrics, involving telomeric regions of 13p and q arms principally and to a lesser degree those of 1p, 16p and 16q arms. These non-random rearrangements paralleled the appearance of clones with unbalanced karyotypes. In particular, two independent proliferating clones were characterized by a monosomy 13. It is concluded that most chromosome lesions directly induced by heavy ions are hardly compatible with cell survival and thus disappear after a few cell generations. However, surviving cells acquire a de novo chromosome instability leading to the formation of clones with unbalanced karyotypes at late passages.

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TL;DR: The results suggest that acentric fragments are good indicators of exposure to very low doses of radiation, although no dose-effect correlation was observed.
Abstract: Cytogenetic studies were performed in lymphocytes from hospital workers exposed to low doses of radiation (1.6-42.71 mSv). When compared with controls, exposed workers showed a significant increase in structural chromosome-type aberrations, acentric fragments being the most frequent alteration. Our results suggest that acentric fragments are good indicators of exposure to very low doses of radiation, although no dose-effect correlation was observed. The incidence of numerical abnormalities (hyperdiploidy) was significantly increased.

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TL;DR: The RFLP/PCR protocol was successfully applied to the determination of N-ethyl-N-nitrosourea-induced mutations in codon 12 of c-H-ras1 and codon 248 of the p53 tumor suppressor gene in human skin fibroblasts and holds promise for molecular toxicology and epidemiology.
Abstract: In most cases somatic mutations in disease-related genes do not give rise to a functional change of the mutated cell which would allow its isolation or expansion in vitro. Therefore, selection of mutated cells on the basis of an altered phenotype has to be replaced by biochemical separation and detection of the altered sequence of the gene of interest. In the RFLP/PCR (RFLP, restriction fragment length polymorphism) approach of 'genotypic' mutation analysis base pair changes, small deletions and insertions are measured which are located in a restriction enzyme recognition sequence and render this site resistant to cleavage by the corresponding endonuclease. The resistant DNA sequence containing the mutated site is amplified by PCR only after wild-type DNA has been eliminated by restriction digestion. Amplified DNA is directly sequenced or cloned into lambda gt10 and mutants are quantitated by oligonucleotide plaque hybridization. Absolute mutation frequencies are estimated relative to an internal 'mutant standard'. The RFLP/PCR protocol has been developed with mixtures of plasmid constructs containing wild-type inserts of the human c-H-ras1 protooncogene or inserts with base pair changes in an MspI site, a PvuII site and a TaqI site of this gene. As few as 1-5 mutated copies could be rescued from 10(7)-10(9) copies of the corresponding wild-type sequence. The RFLP/PCR protocol was successfully applied to the determination of N-ethyl-N-nitrosourea-induced mutations in codon 12 of c-H-ras1 (MspI site 1695-1698) and codon 248 of the p53 tumor suppressor gene (MspI site 14067-14070) in human skin fibroblasts. The RFLP/PCR approach holds promise for molecular toxicology and epidemiology.

Journal ArticleDOI
TL;DR: Analysis of the mutability of the two tk alleles in various tk heterozygotes of WI-L2-NS reveals a similar pattern to that described previously in heterozygote derived from TK6; 3 times as many mutants were recovered from one tk allele than the other.
Abstract: Two human lymphoblastoid cell lineages derived from the same parental line exhibit markedly different survival and mutational responses to X-irradiation, but not to chemical point mutagens. WI-L2-NS (ATCC CRL 8155) and TK6 (ATCC CRL 8015) both are derived from the original WI-L2 isolate described by Levy et al. (1968). Both lines are near diploid with stable and indistinguishable karyotypes (47,X,Y 13+). However, differences in the extent of heterozygosity of chromosome 17 RFLP markers have been detected in these lines. Relative to TK6, WI-L2-NS and several cell lines subsequently derived from it exhibit enhanced survival after X-ray treatment. This is due partly to a more pronounced shoulder in the dose response curve for WI-L2-NS and partly to a higher D 0 than is observed in TK6. X-ray-induced mutant frequencies also are markedly different. At the hprt locus, the overall magnitude of the response is similar in the two cell lines. However, in TK6, a linear equation appears to be the best fit to the data, as compared to a linear quadratic curve for WI-L2-NS. Induced mutant frequencies at the tk locus in heterozygotes derived from WI-L2-NS are 20–50-fold higher than those seen in TK6 and tk heterozygous derivatives of TK6. Analysis of the mutability of the two tk alleles in various tk heterozygotes of WI-L2-NS reveals a similar pattern to that described previously in heterozygotes derived from TK6; 3 times as many mutants were recovered from one tk allele than the other. A possible explanation for the higher survival and induced mutant frequencies seen in WI-L2-NS and its derivatives is the presence in these lines of an error prone repair system not functioning in TK6.

Journal ArticleDOI
TL;DR: Studies using part of the lacI gene from E. coli to measure assay variables are reported, showing that when assay conditions are carefully controlled, the assay is very reproducible.
Abstract: Since its introduction in 1989, analyses by SSCPA of DNA sequences containing mutations by SSCPA have increased dramatically. While many workers have recognised the utility of the technique, few have examined its limitations. In this paper we report studies using part of the lacI gene from E. coli to measure assay variables. When assay conditions are carefully controlled, the assay is very reproducible. The position and type of mutation have little effect on detection efficiency and changes in sequences 176 and 354 bp in length are detected with comparable efficiencies. Overall detection efficiency is > 90% under most conditions. However, local heating due to excessive power levels, can introduce anomalies.

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TL;DR: Although the question of whether dietary calcium can prevent human colorectal cancer remains to be answered, the data presently available appear promising.
Abstract: Many dietary factors have been studied for their potential in the chemoprevention of human colorectal cancer. From an epidemiological standpoint, there have been many studies linking calcium intake to colon cancer risk. Significant reductions in risk have been shown for the consumption of milk, dietary calcium and dairy products in general. Additionally, there have been numerous studies of calcium and cell proliferation in experimental animals. Supplemental calcium in the diet or drinking water has been reported to decrease the colonic epithelial hyperproliferation induced by bile and fatty acids, enteric resection, a nutritional stress diet, and to suppress induction of the tumor-promotion enzyme ornithine decarboxylase. Calcium has also demonstrated an inhibitory effect on experimental colon carcinogenesis. Mechanisms of calcium inhibition are still speculative, but the “calcium soaps” hypothesis, fatty acid destabilization of cellular membranes, modulation of protein kinase C and K- ras mutations are under investigation. Additionally, numerous clinical studies of calcium modulation of human colonic hyperproliferation in high-risk groups as well as chemoprevention trials of calcium supplementation are currently ongoing. Although the question of whether dietary calcium can prevent human colorectal cancer remains to be answered, the data presently available appear promising.

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TL;DR: The results of these studies suggest that the cytochalasin-B Mn/k assay is a cost-effective, simple and rapid alternative to classical cytogenetic assays for the detection of chemically induced aneuploidy.
Abstract: An in vitro micronucleus assay in low passage Chinese hamster Luc2 cells capable of detecting numerical and structural chromosome changes was developed. Chromosome loss was inferred by indirect visualisation of human CREST antikinetochore antibodies bound to centromeres in chemically-induced micronuclei of cytochalasin-B arrested binucleated cells. The assay was used to evaluate 10 chemicals which had been selected for their known or suspected effects upon various components of the cell-division apparatus. These chemicals were colchicine (COL), vinblastine (VBL), thiabendazole (TBZ), chloral hydrate (CH), thimerosal (TM), diazepam (DZ), pyrimethamine (PYR), hydroquinone (HQ), cadmium chloride (CdCl2) and econazole nitrate (EZ). Mitomycin-C (MMC) was used as a positive control for the induction of micronuclei. 8 of the core chemicals induced micronuclei in Chinese hamster Luc2 cells. 4 of the chemicals (COL, VBL, TBZ, CH) increased levels of micronuclei which were positive for kinetochore antibody labelling and hence chromosome loss. 3 of the chemicals (DZ, PYR, HQ) and the positive control (MMC) increased the levels of Mn which were negative for kinetochore antibody labelling. The results with TM were equivocal and EN was negative. The results of these studies suggest that the cytochalasin-B Mn/k assay is a cost-effective, simple and rapid alternative to classical cytogenetic assays for the detection of chemically induced aneuploidy.

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TL;DR: Findings confirm the importance of age and CE as factors which influence the thioguanine-resistant MF in circulating T-lymphocytes from normal adults.
Abstract: Somatic cell mutant frequencies at the hprt locus of the X-chromosome were measured with the T-lymphocyte cloning technique in healthy human populations. A statistical analysis was performed of assays from 232 individuals (77 males and 155 females) ranging in age from 19 to 80 years. Data from 4 donor groups were compiled: (a) 132 participants in a study of identical and fraternal twins; (b) 17 health care workers studied as part of an assessment of the risks of handling chemotherapeutic drugs; (c) 62 women with benign breast masses; and (d) 21 normal laboratory and office personnel. The relationship between age and mutant frequency ( MF ) was expressed by the equation: In MF = 1.46 + 0.018 age ( P MF increased by about 2% per year. Increases in cloning efficiency ( CE ) reduced the MF , as shown in the equation: In MF = 2.91-1.32 CE ( P CE was significantly related to age ( CE = 0.47-0.002 age, P = 0.038), and the interdependent relationship between MF , age and CE expressed by the equation: In MF = 1.99-1.13 CE + 0.016 age was significant at the P MF in our population, but CE was significantly lower in males ( P CE as factors which influence the thioguanine-resistant MF in circulating T-lymphocytes from normal adults.

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TL;DR: Trans-species, multiple site (particularly common site between species), mutagenic rodent carcinogens are less affected by the influences of polymorphic genes than are chemicals inducing more limited carcinogenic effects.
Abstract: Trans-species, multiple site (particularly common site between species), mutagenic rodent carcinogens are less affected by the influences of polymorphic genes than are chemicals inducing more limited carcinogenic effects. Trans-species carcinogens, therefore, should represent a first priority for attention for human health risk.