Fanconi anemia (FA) is a rare, autosomal recessive disorder characterized by elevated frequencies of chromosome aberrations, hypersensitivity to DNA cross-linking agents and predisposition to cancer. At least 5 complementation groups (FA-A to FA-E) underlie FA and the gene defective in FA-C (FAC) has been cloned. The mouse orthologue, Fac, maps in close proximity to the f locus, on chromosome 13, which codes for the flexed-tail mouse phenotype, raising the possibility that f and Fac are synonymous. If this were the case, flexed-tail mice could be used as mouse models for FA-C to help determine the basic defect and to evaluate clinical intervention and gene therapy. To further characterize the flexed-tail mouse, the frequency of micronuclei (a measure of chromosomal aberrations) induced by mitomycin C (MMC), an alkylating and DNA cross-linking agent, was analyzed in peripheral blood and bone marrow erythrocytes. Although a higher spontaneous micronucleus frequency was seen in flexed-tail mice in comparison to wild-type mice, the sensitivity to MMC was not elevated. This result suggests that f and Fac are different genes and that the flexed-tail mouse is not a model for FA-C.
The potentially protective role of chlorophyllin, the sodium and copper salt of chlorophyll a against the initiation and promotion stages in carcinogenesis was studied by in vitro short-term assays. Chlorophyllin showed a dose-dependent suppressive effect on 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1)-induced umu C gene expression of Salmonella typhimurium (TA 1535/pSK 1002) in the presence of metabolizing enzyme mixture. The similar inhibitory effect of chlorophyllin was detected in mitomycin C (MMC)-dependent umu C gene expression in the absence of metabolizing enzyme mixture. Furthermore chlorophyllin also exhibited a dose-dependent inhibition on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity of 3T3 fibroblast cells at the same concentrations. However, when chlorophyll a isolated from Japanese tea leaves was applied on the same assay systems as a comparative experiment, chlorophyll a showed much weaker activity compared with that of chlorophyllin. The significance of this finding is discussed from the viewpoint of the protective role of chlorophyllin against carcinogenesis.
2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which is a heterocyclic amine found in food and the potent mutagen in S. typhimurium TA98, was examined for its genotoxic potential using lacI transgenic mice (Big Blue®, C57BL/6N lineage). Female mice, at 7 weeks of age, were given a diet containing 0.03% MeIQ for 1, 4 and 12 weeks, and mutant frequencies (MF) were analyzed in the bone marrow, liver, forestomach, colon and heart. The MF increased in a feeding period-dependent manner. Relative to untreated mice, the MF after a 12-week-feeding of MeIQ was 38 times higher in the colon, 5.8 times higher in the bone marrow, 4.6 times higher in the liver, and 2.6 times higher in the forestomach. No increase in MF was detected in the heart, where no tumors develop.
There is a strong motivation to develop QSAR models for toxicity prediction for use in screening, for setting testing priorities, and for reducing reliance on animal testing. Decisions must be made daily by toxicologists in governments and industry to direct limited testing to the most urgent public health problems, and to direct the types of chemical synthesis and product development efforts undertaken. This need has motivated attempts to construct general QSAR models (e.g., for rodent carcinogenicity), not tailored to congeneric series of chemicals. These various attempts have provided interesting and important scientific evidence; however, they have also shared a limited overall performance. The goal of this paper is to illustrate, by two unrelated actual examples of QSARs for mutagens and carcinogens, some fundamental problems relative to the application of general QSAR approaches to noncongeneric chemicals. Both examples consider data sets that are noncongeneric in a chemical structure and mechanism of action sense: in the first case, a mean mutagenic potency defined as an average over multiple genetic toxicity endpoints, and, in the second case, the NTP two-sexes, two species rodent carcinogenicity bioassay results for 280 carcinogens and noncarcinogens. The problems encountered with the QSAR analyses of these two cases indicate that a successful approach to the problem of QSAR modeling of noncongeneric data will need to consider the multidimensional nature of the problem in both a chemical and a biological sense. Since different chemical classes represent largely independent action mechanisms, some means for extracting local QSARs for constituent classes will be necessary. Alternatively, a general QSAR derived for a noncongeneric data set will need to be scrutinized and decomposed along chemical class lines in order to establish boundaries for application and confidence levels for prediction.
1,2-Dimethylhydrazine (DMH) was administered to both genders of mice and rats by oral gavage for 3 days. Twenty-four hours later, an assessment of the incidence of micronucleated cells was made in the bone marrow and sections of the gastrointestinal tract. An increase in micronucleated cells was observed in the colon of both genders of both species of rodent. Negative responses were observed in the forestomach, stomach, duodenum, intestine of both species. The bone marrow micronucleus assays were essentially negative, but the absence of a precise definition of the MTD precludes a definitive conclusion from being drawn. These results are consistent with the selective carcinogenicity of DMH to the colon of the rodent GI-tract. DMH is also known to be carcinogenic to rat and mouse liver and, although it is known to induce micronuclei in the hepatocytes of rats, no such data exist for the mouse. Consequently, mice were administered DMH on 13 successive days, followed by 2/3 partial hepatectomy and assessment of micronucleated hepatocytes. A strong positive liver micronucleus assay response was observed. Thus, DMH selectively induces micronuclei in the colon and liver of rats and mice, consistent with its carcinogenicity to these two tissues. No qualitative differences between the genders was observed in any of the assays. These results indicate that the assessment of genetic toxicity in rodents should not rely solely on assays made in bone marrow.
Initial analyses of the lung tumors seen in rats exposed for their lifetime to elevated levels of the emissions of diesel engines suggested that they were due to powerful mutagens and carcinogens (PAHs, nitro PAHS) adsorbed onto the diesel particles. However, further studies showed that carcinogenicity occurred only under conditions that resulted in impaired lung clearance (‘overloading’) leading to inflammatory reactions and other pathologic sequelae. These observations together with the findings that carbon black, a model for diesel particles devoid of organic mutagens and carcinogens, also induced lung cancers under conditions of overloading led to the suggestion that the cancers resulted from a non-genotoxic mechanism. However, the further finding that inert particulate carcinogens devoid of organics, induced mutations has led to a re-evaluation of the role of mutations in lung carcinogenesis caused by particles and the relevance of the rat model to humans. This is especially timely as epidemiological studies suggest that humans may develop lung cancers following occupational exposure to diesel emissions by a mechanism unlikely to involve lung overloading. Finally, the recent recognition that environmental PM-10 (respiratory size particles) may be responsible for a significant portion of human morbidity and mortality, ensures that the health effect of diesel emissions will continue to receive scrutiny as they contribute to the PM-10 load.
Indoor air pollution has now been recognized as a potentially important problem for public health, since people spend most of their day in closed environments. Incense burning is possibly associated with elevated risks of leukemia and brain tumor in children from the epidemiological studies. Thus, evaluation of the genotoxicity of smoke condensates from incense burning is needed. We examined the genotoxicity of incense smoke condensates (ISC) using the Ames test in S. typhimurium strains with different mutagenic specificity and level of metabolic enzyme, the SOS chromotest in E. coli PQ37, and sister chromatid exchange assay in Chinese hamster ovary cells (SCE/CHO). The genotoxicity of environmental tobacco smoke condensates (TSC) was also evaluated by the three assays to compare with the genotoxicity of ISC. ISC showed a positive response in TA98, but not in TA100. It suggested that ISC only contained frame shift mutagens. The mutagenicity of ISC in both strains of TA98NR with deficient nitroreductase and TA98/1,8-DNP6 with deficient O-acetyltransferase was markedly decreased compared to that in TA98 strain. However, the mutagenicity was enhanced in YG1024 with overexpression of O-acetyltransferase activity. Thus, nitroarenes seemed to be responsible in part for the mutagenicity of ISC. Interestingly, all of the four ISC and two TSC samples showed a dose-dependent genotoxic response in the SOS chromotest with E. coli PQ37 but a low SCE induction of those samples were observed in CHO cells. When the genotoxicity was analyzed based on the condensates per one gram of original samples, the genotoxicity of two TSC condensates in prokaryotic cells was higher than that of four ISC samples except for the genotoxicity of TSC-2 in TA98 strain. However, the genotoxicity of certain ISC in eukaryotic cells based on the SCE/CHO assay was higher than that of TSC. To compare the covalent binding of DNA reactive intermediates of ISC and TSC to S. typhimurium TA98, the DNA adducts were evaluated by the 32P-postlabeling method with butanol extraction version. Similar diagonal radioactive zone (DRZ) was observed between ISC and CSC. However, DNA adduct levels induced by TSC were much greater than that of ISC.
Monomeric acrylamide was tested for its potential to induce aneuploidy in spermatocytes and bone marrow cells of mice. For this purpose, chromosomes from metaphase spreads were counted semi-automatically. In both test systems, cell proliferation was monitored, determining the meiotic index of spermatocytes and the average generation time of bone marrow cells after BrdU incorporation, respectively. No indications could be seen for different sensitivity of somatic and germinal cells towards acrylamide. With a dose of 120 mg/kg, the chemical caused cell cycle delay in both germ line and somatic cells. There was diverging response with respect to the balance of hypo- and hyperploidy. While the percentage of chromosome loss was significantly elevated in both test systems, acrylamide treatment did not increase the frequency of hyperploid cells. Interpreting these results on the basis of conventional test protocols, acrylamide should not be considered as an aneugen. The conservative approach, however, may be inadequate for the detection of aneugenic mechanisms different from non-disjunction.
Chromosomal aberrations (CA) were studied in the peripheral blood lymphocytes of 85 healthy male volunteers from Barcelona (Catalonia, Spain). The effect that factors such as age, life style, work exposure and medical treatment had on the cytological endpoints was studied by means of a Poisson regression model. The results obtained indicate a significant positive relationship between the age of the subjects analyzed and the total of chromosome-type aberrations. With respect to the other variables analyzed, a positive association was found between the frequent consumption of analgesics and the incidence of chromatid-type aberrations.
Exposure of cells to low doses of radiation or chemicals renders them more resistant to higher doses of these agents. This phenomenon, termed adaptive response, was studied in quiescent human keratinocytes exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The cells were adapted with 2.5 nM MNNG for 60 min and challenged immediately thereafter with 2.5 microM MNNG for 30, 45 or 60 min. Clonogenic survival studies revealed that adapted cells were more resistant to the subsequent challenge treatment (up to 30% higher survival) than unadapted cells. In addition, formation of DNA strand breaks was lower in adapted cells. We monitored poly-ADP-ribosylation activity during expression of the adaptive response both at the substrate as well as the product level. NAD+ utilization in adapted and non-adapted cells exposed to the high dose of MNNG was similar, but recovery from NAD+ depletion was faster in low-dose pretreated cells. Induction of poly(ADP-ribose) formation was more than 2 times higher in low-dose adapted cells and this was associated with the formation of a distinct class of ADP-ribose polymers, i.e., branched polymers. These polymers exhibit a very high binding affinity for histones and can displace them from DNA. Elevated levels of poly(ADP-ribose) and, particularly, synthesis of branched polymers may play a critical role in low-dose adaptation.
In vivo sister chromatid exchange (SCE) and chromosome aberrations (CA) were carried out for six salicylic acid derivatives in bone marrow cells of mice. Six salicylic acid derivatives, namely acetyl salicylic acid (aspirin), salicylic acid, salicylamide, sodium salicylate, diflunisal and niclosamide, were used for these experiments. Drugs were administered both intraperitoneally (i.p.) and orally by gavage. Out of these six salicylic acid derivatives tested, only diflunisal and niclosamide showed genotoxicity as measured by both SCE and CA assays. Acetyl salicylic acid and sodium salicylate showed weak genotoxicity as measured by SCE and CA, respectively, only at the highest dose tested.
A multibiomarker approach based on the study of toxicity mechanisms at both genetic and metabolic levels has been applied to Fenarimol. With regard to genotoxicity, particular attention was given to assays for chromosomal aberration and micronuclei; clastogenic potential was assessed in human peripheral blood lymphocytes in vitro, while the induction of micronuclei was studied in male CD1 mouse bone marrow polychromatic erythrocytes (PCE). Fenarimol did not induce any significant increase in chromosomal aberrations in vitro, either in the presence or absence of rat liver S9 fraction. In contrast, a significant dose-related increase in micronucleated PCEs, up to 4-fold above the control level at a single dose of 75 mg/kg b.w., was observed 24 h after treatment. Using selective biochemical markers of effect Fenarimol was found to induce CYP 2B1 isoforms in liver, kidney and lung microsomes of Swiss Albino CD1 male and female mice, as shown by the significant increase in specific 2B1-probe pentoxyresorufin O-dealkylase activity. On the contrary, CYP 3A, probed by N-demethylation of aminopyrine, were only induced in the liver. Results were corroborated by by means of Western immunoblotting using rabbit polyclonal antibodies anti-CYP 2B1 and 3A. Northern blotting analysis with CYP 2B1 and 3A cDNA biotinylated probes showed that the expression of such isoforms is regulated at mRNA level. Taken as a whole, these data indicate the possible (mutagenic) cotoxic/cocarcinogenic and promoting potential of this fungicide.
The present in vitro and in vivo experiments were undertaken to clarify the genotoxic potential of the hydroxyanthrachinone aloeemodin which can be found in different plant derived products for therapy of constipation. The results demonstrate that aloeemodin is able to induce mutagenic effects in vitro. Positive results were obtained in the chromosomal aberration assay with CHO cells, as well as in the Salmonella reverse mutation assay (frameshift mutations in strains TA 1537, TA 1538 and TA 98). No mutagenic potential of aloeemodin, however, was observed in the gene mutation assay with mammalian cells in vitro (HPRT assay in V79 cells). Each assay was performed in the presence and absence of an extrinsic metabolic activation system (S9-mix). In in vivo studies (micronucleus assay in bone marrow cells of NMRI mice; chromosome aberration assay in bone marrow cells of Wistar rats; mouse spot text [DBA/2JxNMRI]) no indication of a mutagenic activity of aloeemodin was found. Information about a possible reaction of aloeemodin with DNA was derived from an in vivo UDS assay. Hepatocytes of aloeemodin-treated male Wistar rats did not show DNA damage via repair synthesis. All these data suggest that aloeemodin is able to interact with DNA under certain in vitro conditions. However, in vivo the results that were negative did not indicate a genotoxic potential. Therefore, it may be assumed that a genotoxic risk for man might be unlikely.
The herbicide trifluralin was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister-chromatid exchanges (SCE), chromosome aberrations (CA) and micronuclei (MN) were scored as genetic endpoints. To detect eventual metabolic modification in the genotoxicity of this herbicide, the cultures for SCE and MN demonstration were also treated with S9 fraction. From our results we can conclude that trifluralin was able to exert a weak cytotoxic effect, reducing both the proliferative rate index (PRI) and the cytokinesis block proliferation index (CBPI), and also to induce a slight but statistically significant increase in the frequency of SCE. Under our conditions of testing, no genotoxic effects of trifluralin were observed in the CA and MN assays.
Using the Salmonella/microsome assay, the antimutagenic effects of specific components of the extracts from eggplant fruits were investigated. The eggplant fruit juice exhibited an antimutagenic activity against 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) induced mutagenicity. In some of the fractions extracted with several organic solvents (acetone, petroleum ether, ethyl acetate, and methanol), the activity was recognized. No mutagenicity or toxicity for Salmonella typhimurium TA98 in the presence of S9 mixture was observed with any of the extracts. It is suggested that there are multiple components of the activities that exist in the eggplant fruit. We isolated lutein from the 84% methanol (methanol/water, v/v) layer, pheophorbide or chlorophyllide from the 70% methanol layer and tannins containing sugar-moieties from the water layer. Pheophytin a and b, Mg-free derivatives of chlorophyll a and b, were isolated from the petroleum ether layer as possible antimutagens. The pheophytin a with S9 mix inhibited by 30–40% the mutagenicity of Trp-P-2.
Water samples were collected from the river Ganga at Narora (U.P.). High performance liquid chromatography analysis of water samples by the liquid extraction procedure indicated the presence of several pesticides such as DDT, α-BHC, aldrin, endrin and dieldrin at concentrations of 1.36, 1.38, 0.95, 0.61 and 0.41 ppb, respectively. The organophosphorus pesticides such as dimethoate and methyl parathion also appear to be present at concentrations of 0.20 and 0.41 ppb, respectively. The XAD water concentrates and liquid-liquid extracted water samples were assayed for mutagenic potential by the Ames Salmonella/microsome test. The test samples exhibited a significant degree of mutagenicity with TA102, TA100 and TA98 strains both in the presence and absence of DNA repair defective mutants, recA, lexA and polA of E. coli was observed as compared to their wild-type countepart in the presence of XAD water concentrates.
The radiotherapy treatment of human cancer is often limited by the side effects and complications induced in normal surrounding tissues. The use of therapeutic strategies that could protect normal tissues while permitting the death of malignant neoplasm would be advantageous. Some studies have suggested that the amino acid glutamine (GLN) can serve as a conditionally essential nutrient in patients in a catabolic condition. The objective of this study was to evaluate the possible radioprotection of GLN on the frequency of chromosomal aberrations, number of metaphases with chromosomal aberrations and mitotic index in bone marrow cells of Rattus norvegicus. In this in vivo test system, GLN was administered by gavage at concentrations of 300 and 600 mg/kg body weight, in acute treatments, 30 min or 24 h before exposure to 3 Gy of whole-body gamma radiation. The results obtained in these experiments showed that GLN did not alter significantly the frequency of chromosome aberrations induced by gamma radiation under the experimental conditions used in the present study.
NNK is a potent environmental carcinogen generated during tobacco processing and smoking. The carcinogenic response to tobacco smoking is modulated by nutritional factors. In this study, liver microsomes from phenobarbital and β-naphthoflavone-treated or control hamsters were used to assay the mutagenicity (Salmonella typhimurium TA1535) of NNK. Western analysis of these microsomal preparations revealed an increased expression of protein recognized by polyclonal antibodies specific for P-450 1A2 in β-naphthoflavone-induced microsomes and P-450 2B1/2B2 in phenobarbital-induced microsomes. Both inducers significantly increased the mutagenicity of NNK. Metabolism of NNK by the three microsomal preparations was compared. Metabolites formed by methyl-hydroxylation of NNK by microsomes from control animals were significantly greater than those formed by α-methylene hydroxylation. Phenobarbital treatment had the greatest effect on α-methylene hydroxylation while β-naphthoflavone had the greatest effect on methyl hydroxylation. The antimutagenic action of the polyphenolic compounds ellagic acid, esculetin and propyl gallate correlated with an inhibition of the metabolism of NNK. There were, however, differences in the effects of these compounds on specific pathways of NNK metabolism depending upon the microsomal enzyme induction treatment. This suggests that phenolic compounds have selective affinity for specific P-450 isozymes activating NNK.
Genotoxic evaluation of a commonly used progestogen, norethisterone acetate, was undertaken using a combination of short-term in vitro and in vivo assays. The clastogenic potentiality of norethisterone acetate was evident from the chromosome aberrations and sister chromatid exchanges induced both with and without S9 mix in cultured human lymphocytes and also from the increased frequency of micronuclei formation and sister chromatid exchanges in mice. However, in the Ames Salmonella assay, both with and without S9 mix and in host-mediated assay, norethisterone acetate was unable to cause any significant increase/decrease in the His+ revertants/plate.
We examined the binding of various steroid hormones to DNA in vitro by means of 32P-postlabeling. Seventeen steroid hormones and cholesterol (CS) were incubated with human liver DNA at 37°C for 1 h under aerobic conditions in the absence of catalysis. The reaction mixtures were analyzed by the nuclease P-1 version of 32P-postlabeling. The results showed that cortexolone (CX), prednisolone (PS), cortisone (CN), cortisol (CL), tetrahydrocortisol (TC), corticosterone (CC), 11-deoxycorticosterone (DC), dexamethasone (DX), dihydrocortisol (DL), and aldosterone (AL) covalently bound with DNA. However, progesterone (PG), 17α-hydroxyprogesterone (HG), estrone (E1), estradiol (E2), estriol (E3), testosterone (TS), cortol (CR) and the original compound for biosynthesis, CS, did not form adducts. In absence of DNA, the steroids themselves did not give rise to any spot on TLC under the same conditions. The dose-responses of DNA binding by DC, DL, CC, CL and CN were linear. The relative adduct labeling of reactive steroids at a concentration of 2 mM were as follows: 68.8 (CX), 53.2 (PS), 39.6 (CN), 29.9 (CL), 20.9 (TC), 12.9 (CC), 12.3 (DC), 7.5 (DX), 4.7 (DL), 1.2 (AL) adducts per 108 nucleotides. Reactive and nonreactive steroids were distinguishable by the presence or absence of the carbonyl group (-CO-CH2OH) at carbon seventeen (C17) of the cholesterol skeleton. This implies that the electrophilic carbonyl or a neighboring group perhaps involved in the formation of covalent bond with DNA. To investigate the nature of target base(s) of these DNA reactive steroids, mononucleotides of all four bases of DNA were reacted with CN, CL, CC and cochromatographed with the obtained spots of DNA reactions. The results of which stated that these steroids and guanine reaction gave the same spots as observed in DNA reaction, indicating guanine is the main target of these DNA reactive steroids. Hep G2 human hepatocellular carcinoma cells were used as an alternative model. Although nine steroids (CL, DL, TC, PS, DX, PG, E2, TS, CR) did not react with intracellular DNA under our experimental conditions, our findings suggested that some hormonal steroids can form covalent DNA adducts in vitro.
