Mutagen Methyl Methanesulfonate Research Articles

A cytogenetic study of the antimutagenic potential of herbal infusions from Matricaria chamomilla L. and Achillea millefolium L. (Fam. Asteraceae)

Due to increase of hazardous factors in the environment, it becomes relevant to search for effective protectors of natural origin for the correction of toxic and genetic effects induced by xenobiotics. Using the test for count chromosomal abnormalities in cells of root germinal meristem of barley seeds, the mutagenic and antimutagenic activity of infusions from chamomile (Matricaria chamomilla) and yarrow (Achillea millefolium) were studied. The studied infusions with various preparation methods (concentrated, diluted and phyto tea) did not show mutagenic activity. The frequency of structural mutations was at the level of negative control (distilled water). The ability of yarrow and chamomile infusions to reduce MMS-induced mutagenesis has been established. For direct and reverse treatment of seeds with diluted infusions or herbal tea of medicinal plants and mutagen methyl methanesulfonate (MMS, positive control) a statistically significant decrease of MMS-induced was observed (p<0.05). Moreover, the level of inhibition of the mutation process depended on the sequence of exposure to infusions and mutagen, as well as the type of infusion. The effectiveness of the antimutagenic effect of the studied infusions was evaluated by the reduction factor. The reduction factor in the infusion of diluted chamomile and chamomile tea was 67.0% and 62.0%, respectively, which indicates the ability to inhibit MMS-induced mutagenesis by more than 60% from Matricaria chamomilla infusions. The results indicate a strong antimutagenic effect of diluted infusions of chamomile. The magnitude of the reduction factor in yarrow infusions indicates the ability of infusions from Achillea millefolium to inhibit MMS-induced mutagenesis by 40-50% with preliminary exposure to barley to MMS. The reduction factor subsequent to the effect of infusions after MMS was 45-50%. The results obtained indicate the presence of antimutagenic activity in infusions of chamomile and yarrow, due to the presence of biologically active substances of various nature in plants of these species. Key words: biologically active substances, medicinal plants, mutagenesis, antimutagenic activity, chromosomal aberrations.

An Adaption of Human-Induced Hepatocytes to In Vitro Genetic Toxicity Tests.

Metabolic activation is essential in standard in vitro genotoxicity test systems. At present, there is a lack of suitable cell models that can express the major characteristics of liver function for predicting substance toxicity in humans. Human-induced hepatocytes (hiHeps), which have been generated from fibroblasts by lentiviral expression of liver transcription factors, can express hepatic gene programs and can be expanded in vitro and display functional characteristics of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Our purpose was to investigate whether hiHeps could be used as a more suitable model for genotoxicity evaluation of chemicals. Therefore, a direct mutagen, methylmethanesulfonate (MMS), and five promutagens [2-nitrofluorene (2-NF), benzo[a]pyrene (B[a]P), aflatoxin B1, cyclophosphamide and N-nitrosodiethylamine] were tested by the cytokinesis-block micronucleus test and the comet assay. Results from genotoxicity tests showed that the micronucleus frequencies were significantly increased by all of the six clastogens tested. Moreover, MMS, 2-NF and B[a]P induced significant increases in the % Tail DNA in the comet assay. In conclusion, our findings from the preliminary study demonstrated that hiHeps could detect the genotoxicity of indirect carcinogens, suggesting their potential to be applied as an effective tool for in vitro genotoxicity assessments.

Protective effects of Solanum cernuum extract against chromosomal and genomic damage induced by methyl methanesulfonate in Swiss mice.

Solanum cernuum Vell is a Brazilian shrub or small tree, restricted to Southeast states of the country. The leaves are commercialized as "panacéia" and indicated for the treatment of urinary disorders, gonorrhea, scabies, skin diseases and as desobstruent, diuretic and antiarrhythmic. The hydroalcholic extract is active in the treatment of gastric ulcer. The aim of this study was to evaluate the genotoxic and antigenotoxic potential of S. cernuum hydroalcoholic extract (SC) in Swiss mice by micronucleus and comet assays. The animals were treated by gavage with the doses of 500, 1000 and 2000mg/kg body weight (b.w.). For antigenotoxicity assessment, the doses of 15, 30, 60, 120 and 240mg/kg b.w SC were administered simultaneously with the mutagen methyl methanesulfonate (MMS, 40mg/kg b.w., i.p.). The results showed that the SC was not genotoxic in both micronucleus and comet assays. On the other hand, the treatment with the lowest dose of SC (15mg/kg b.w.) plus MMS showed a statistically significant reduction in the frequency of micronuclei compared to treatment only with MMS. For the comet assay, significant reduction in extensions of DNA damage was observed in all treatments with SC combined with MMS in comparison with only MMS. The antigenotoxic activity observed for the SC may be due to the antioxidant potential of the compounds present in the extract such as guanidine alkaloids and flavonoids.

Antimutagenic Thio Compounds from Sisymbrium officinale

Glucoputranjivin (1) and isopropyl isothiocyanate (2) were isolated from an aqueous dry extract of Sisymbrium officinale and were identified by spectroscopic analysis. The antimutagenic activity of these compounds was evaluated in a bacterial reverse mutation assay using E. coli WP2, WP2uvrA, and WP2uvrA/pKM101 strains, in comparison with the extract. In the absence of the exogenous metabolic activation system S9, the thio compounds exerted antimutagenic activity against the direct-acting mutagen methyl methanesulfonate, in all strains. In the presence of S9, both thio compounds were active against the indirect mutagens 2-aminoanthracene, in WP2uvrA, and 2-aminofluorene, in WP2. The antimutagenicity seems to be due to specific mechanisms, such as the induction of the adaptive response or the excision repair system. Conversely, the inhibition of the CYP450-mediated activation of mutagens was not supported by the present results. An antimutagenic effect was also observed for the S. officinale aqueous extract against the arylamines 2AA and 2AF, but not against MMS. These results suggest that both thio compounds are involved in the antimutagenicity of S. officinale. The antimutagenicity of glucosinolate 1 is reported for the first time.

Polar extracts from (Tunisian) Acacia salicina Lindl. Study of the antimicrobial and antigenotoxic activities

BackgroundMethanolic, aqueous and Total Oligomer Flavonoids (TOF)-enriched extracts obtained from the leaves of Acacia salicina 'Lindl.' were investigated for antibacterial, antimutagenic and antioxidant activities.MethodsThe antimicrobial activity was tested on the Gram positive and Gram negative reference bacterial strains. The Mutagenic and antimutagenic activities against direct acting mutagens, methylmethane sulfonate (MMS) and 4-nitro-o-phenylenediamine (NOPD), and indirect acting mutagens, 2-aminoanthracene (2-AA) and benzo[a]pyrene (B(a)P) were performed with S. typhimurium TA102 and TA98 assay systems. In addition, the enzymatic and nonenzymatic methods were employed to evaluate the anti-oxidative effects of the tested extracts.ResultsA significant effect against the Gram positive and Gram negative reference bacterial strains was observed with all the extracts. The mutagenic and antimutagenic studies revealed that all the extracts decreased the mutagenicity induced by B(a)P (7.5 μg/plate), 2-AA (5 μg/plate), MMS (1.3 mg/plate) and NOPD (10 μg/plate). Likewise, all the extracts showed an important free radical scavenging activity towards the superoxide anion generated by the xanthine/xanthine oxidase assay system, as well as high Trolox Equivalent Antioxidant Capacity (TEAC), against the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)+• radical. TOF-enriched extract exhibited the highest protective effect against free radicals, direct acting-mutagen and metabolically activated S9-dependent mutagens.ConclusionsThe present study indicates that the extracts from A. salicina leaves are a significant source of compounds with the antimutagenic and antioxidant activities, and this may be useful for developing potential chemopreventive substances.

Single-Molecule DNA Repair in Live Bacteria

Cells rely on sophisticated DNA repair mechanisms to maintain integrity of a genome that is under constant attack by mutagenic chemicals, ionizing radiation, or UV light. Single-molecule fluorescence microscopy allows, for the first time, direct observation of DNA repair in living bacteria. This in vivo approach enables a quantitative description of the chain of events within an entire repair pathway, studying control, timing, and coordination during the initial damage response, the search for damaged sites, and the repair protein function.Using photoactivation, super-resolution localization, and tracking we follow the movement of individual molecules of DNA Polymerase I, a protein essential for DNA repair synthesis and lagging strand replication in E.coli. Consistent with early reports, we measure 300 copies of Pol1 per cell; the molecules have a spatial distribution reflecting that of the bacterial chromosome. In cells without DNA damage, most Pol1 molecules move with an apparent diffusion coefficient of 1.75 um2/s, consistent with simulations of unhindered Brownian motion within a cell. We observe significant displacement of Pol1 molecules between consecutive 15-ms frames, setting an upper limit on the DNA residence time in a facilitated diffusion model that combines 1-D diffusion on DNA with a 3-D random walk in the cytoplasm.In contrast, in cells with DNA damage by mutagen methyl-methanesulfonate (MMS), we clearly observe stationary Pol1 molecules, and individual tracks showing binding and dissociation events; the fraction of bound-to-free Pol1 scales with the damage extent. These observations suggest that stationary Pol1 molecules are performing repair synthesis to fill short single-stranded DNA gaps created by the base excision repair (BER) machinery. Current work focuses on the real-time DNA damage response as a function of protein concentration and binding activity, and examining additional repair proteins to build a comprehensive model of the BER pathway in vivo.

Genotoxic damage in polychaetes: A study of species and cell-type sensitivities

The marine environment is becoming increasingly contaminated by environmental pollutants with the potential to damage DNA, with marine sediments acting as a sink for many of these contaminants. Understanding genotoxic responses in sediment-dwelling marine organisms, such as polychaetes, is therefore of increasing importance. This study is an exploration of species-specific and cell-specific differences in cell sensitivities to DNA-damaging agents in polychaete worms, aimed at increasing fundamental knowledge of their responses to genotoxic damage. The sensitivities of coelomocytes from three polychaetes species of high ecological relevance, i.e. the lugworm Arenicola marina, the harbour ragworm Nereis diversicolor and the king ragworm Nereis virens to genotoxic damage are compared, and differences in sensitivities of their different coelomic cell types determined by use of the comet assay. A. marina was found to be the most sensitive to genotoxic damage induced by the direct-acting mutagen methyl methanesulfonate (MMS), and showed dose-dependent responses to MMS and the polycyclic aromatic hydrocarbon benzo( a)pyrene. Significant differences in sensitivity were also measured for the different types of coelomocyte. Eleocytes were more sensitive to induction of DNA damage than amoebocytes in both N. virens and N. diversicolor. Spermatozoa from A. marina showed significant DNA damage following in vitro exposure to MMS, but were less sensitive to DNA damage than coelomocytes. This investigation has clearly demonstrated that different cell types within the same species and different species within the polychaetes show significantly different responses to genotoxic insult. These findings are discussed in terms of the relationship between cell function and sensitivity and their implications for the use of polychaetes in environmental genotoxicity studies.

Antimutagenic activity of cashew apple (Anacardium occidentale Sapindales, Anacardiaceae) fresh juice and processed juice (cajuína) against methyl methanesulfonate, 4-nitroquinoline N-oxide and benzo[a]pyrene

Cashew apple juice (CAJ), produced from the native Brazilian cashew tree (Anacardium occidentale), and has been reported to have antibacterial, antifungal, antitumor, antioxidant and antimutagenic properties. Both the fresh unprocessed juice and the processed juice (cajuína in Portuguese) has been shown to consist of a complex mixture containing high concentrations of anacardic and ascorbic acids plus several carotenoids, phenolic compounds and metals. We assessed both types of juice for their antimutagenic properties against the direct mutagens methyl methanesulfonate (MMS) and 4-nitroquinoline-N-oxide (4-NQO) and the indirect mutagen benzo[a]pyrene (BaP) using pre-treatment, co-treatment and post-treatment assays with Salmonella typhimurium strains TA100, TA102, and TA97a. In pre-treatment experiments with strains TA100 and TA102 the fresh juice showed high antimutagenic activity against MMS but, conversely, co-treatment with both juices enhanced MMS mutagenicity and there was an indication of toxicity in the post-treatment regime. In pre-, co-, and post-treatments with TA97a as test strain, antimutagenic effects were also observed against 4-NQO and BaP. These results suggest that both fresh and processed CAJ can protect the cells against mutagenesis induced by direct and indirect mutagens.

Effect of Safranal, a Constituent of Crocus sativus (Saffron), on Methyl Methanesulfonate (MMS)–Induced DNA Damage in Mouse Organs: An Alkaline Single-Cell Gel Electrophoresis (Comet) Assay

The influence of safranal, a constituent of Crocus sativus L. stigmas, on methyl methanesulfonate (MMS)-induced DNA damage was examined using alkaline single-cell gel electrophoresis (SCGE), or comet, assay in multiple organs of mice (liver, lung, kidney, and spleen). NMRI mice were divided into five groups, each of which contained five mice. The animals in different groups were received the following chemicals: physiological saline (10 mL/kg, ip), safranal (363.75 mg/kg, ip), MMS (120 mg/kg, ip), safranal (72.75 mg/kg, ip) 45 min prior to MMS administration, and safranal (363.75 mg/kg, ip) 45 min prior to MMS administration. Mice were sacrificed about 3 h after the administration of direct mutagen MMS, safranal, or saline, and the alkaline comet assay was used to evaluate the influence of safranal on DNA damage in different mouse organs. Increase in DNA migration was varied between 9.08 times (for spleen) and 22.12 times (for liver) in nuclei of different organs of MMS-treated mice, as compared with those of saline-treated animals (p < 0.001). In control groups, no significant difference was found in the DNA migration between safranal- and saline-pretreated mice. The MMS-induced DNA migration in safranal-pretreated mice (363.75 mg/kg) was reduced between 4.54-fold (kidney) and 7.31-fold (liver) as compared with those of MMS-treated animals alone (p < 0.001). This suppression of DNA damage by safranal was found to be depended on the dose, and pretreatment with safranal (72.75 mg/kg) only reduced DNA damage by 25.29%, 21.58%, 31.32%, and 25.88% in liver, lung, kidney, and spleen, respectively (p < 0.001 as compared with saline-treated group). The results of the present study showed that safranal clearly repressed the genotoxic potency of MMS, as measured by the comet assay, in different mouse organs, but the mechanism of this protection needs to be more investigated using different in vitro system assays and different experimental designs.

Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal-plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Deltacpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Deltacpku80 strain, a significant increase over the 2-5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica.

Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents

In the present study, the chemical composition of Origanum compactum essential oil was determined by gas chromatography and mass spectrometry, and its mutagenic and antimutagenic activities were investigated by the somatic mutation and recombination test (SMART) in Drosophila melanogaster. No significant increase in the number of somatic mutations was observed with the essential oil tested using both the standard (ST) and high bio-activation (HB) cross. In order to investigate the antimutagenic effect of the essential oil, we have tested the effect on the indirect-acting mutagen urethane (URE), as well as the direct-acting mutagen methyl methanesulfonate (MMS). O. compactum essential oil showed a strong inhibitory effect against URE-induced mutagenicity, especially with the HB cross. However, only a weak inhibitory effect on the mutagenicity induced by MMS was observed. These results suggest that the detected antimutagenicity could be mediated by an inhibitory effect on metabolic activation. The essential oil was fractionated to identify the components responsible of the suppressing effect detected. Seven fractions were obtained: two of them showed the most potent inhibitory effect against URE-induced mutagenicity and were further fractionated. The sub-fractions obtained from the second chromatographic fractionation were tested for their antimutagenic activity, together with carvacrol and thymol. The highest antimutagenic effect obtained with the sub-fractions was similar to the effect of the crude essential oil, as well as to the effect of carvacrol alone. These results suggest the absence of a synergic antimutagenic effect between the components of O. compactum essential oil and indicate that carvacrol was the most active oil component.

Anticancer Effects of Four Varieties of Muscadine Grape

The initiating event in carcinogenesis is a somatic mutation. During progression of the disease, additional mutations accumulate as the transformed cells develop the ability to proliferate and metastasize. These mutations can be produced by reactive oxygen species (ROS) generated through metabolism, or environmental insult. Metastasis involves tissue degradative enzymes, many of which are members of the matrix metalloproteinase family. Hence, substances that can neutralize ROS, inhibit mutagenesis, or block activity of the matrix metalloproteinases should prove to be anticarcinogenic. This study was performed to evaluate the possible anticarcinogenic characteristics of muscadine grapes. These grow wild in the southeast United States and have not been subjected to extensive breeding, as have most commercially cultivated fruits. The extracts tested were from pomace remaining after wine production. This is usually discarded, but the results obtained in this study indicate that pomace water extracts could be used as sources for purification of anticarcinogenic compounds. Four varieties of muscadine grape were tested for their abilities to affect mutagenesis by the metabolically activated carcinogen 2-aminoanthracene. Each extract was also assayed for antioxidant activity and for its ability to inhibit activity of matrix metalloproteinases-2 and -9. Each of the four extracts showed significant inhibition of 2-aminoanthracene mutagenesis, high antioxidant activity, and the ability to inhibit activities of both metalloproteinases, implying that these extracts could be good inhibitors of carcinogenesis. Two of the extracts showed little activity when tested for their effects on mutagenesis by the direct-acting mutagen methyl methanesulfonate.

Drosophila Damaged DNA Binding Protein 1 Contributes to Genome Stability in Somatic Cells

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.

Antimutagenic Activity of Berry Extracts

Plants are proven sources of useful anti-tumor and chemopreventative compounds. Hence, identification of phytochemicals useful in dietary prevention and intervention of cancer is of paramount importance. The initial step in the formation of cancer is damage to the genome of a somatic cell producing a mutation in an oncogene or a tumor-suppressor gene. Fresh juices and organic solvent extracts from the fruits of strawberry, blueberry, and raspberry were evaluated for their ability to inhibit the production of mutations by the direct-acting mutagen methyl methanesulfonate and the metabolically activated carcinogen benzo[a]pyrene. Juice from strawberry, blueberry, and raspberry fruit significantly inhibited mutagenesis caused by both carcinogens. Ethanol extracts from freeze-dried fruits of strawberry cultivars (Sweet Charlie and Carlsbad) and blueberry cultivars (Tifblue and Premier) were also tested. Of these, the hydrolyzable tannin-containing fraction from Sweet Charlie strawberries was most effective at inhibiting mutations.

The QDE-3 homologue RecQ-2 co-operates with QDE-3 in DNA repair in Neurospora crassa.

The post-transcriptional gene silencing mechanism in Neurospora crassa, called quelling, was shown to involve the products of three genes termed quelling-defective. A homologue to the qde-3 gene encoding a putative RecQ-type DNA helicase was isolated and was named RecQ-2. Characterisation of the RecQ-2 gene has revealed that it is not involved in quelling, but may co-operate with the qde-3 gene product in a pathway that repairs damage to DNA caused by the chemical mutagens methyl methanesulfonate and N-methyl- N'-nitro- N-nitrosoguanidine. These results indicate that the qde-3 RecQ helicase may have a dual role in N. crassa, either acting alone as an essential component of the quelling mechanism or together with the RecQ-2 RecQ helicase, as part of a process to repair DNA lesions during replication.

Induction of epithelial tumors in Drosophila melanogaster heterozygous for the tumor suppressor gene wts.

The imaginal disk cells of Drosophila have a cell cycle that is very similar to that of mammalian cells. Data concerning factors inducing tumors in these cells may directly relate to the risk of these factors for inducing cancer in humans. One of the genes involved in the regulation of cell cycle control is wts (warts), the Drosophila homolog of the mammalian tumor suppressor gene LATS1. The Drosophila wts mutations are recessive lethal. However, homozygous clones that arise in heterozygous flies in the imaginal disk cells lead to epithelial tumors, spectacular outgrowths visible on the cuticle of the adult. We have treated Drosophila larvae, heterozygous for wts, with the chemical mutagen MMS (methyl methanesulfonate) or with X-rays and measured the appearance of epithelial tumors in the eclosing adult flies. This test is a variation of the well-known Drosophila somatic mutation and recombination test (SMART), where mostly recessive markers have been used leading to visible phenotypes in the eyes and wings of the fly. We show that the sensitivity of this test is far greater than the comparable test system using the recessive eye marker white.

Antimutagenic effects of the mushroom Agaricus blazei Murrill extracts on V79 cells

Agaricus blazei Murrill, a native mushroom in Brazil, has been widely consumed in different parts of the world due to its medicinal power. Its anticarcinogenic activity has been shown in experimental animals, and antimutagenic activity has been demonstrated only in Salmonella. In this work, the mutagenic and antimutagenic activities of mushroom teas of strains AB96/07, AB96/09 and AB97/11 were evaluated in Chinese hamster V79 cells, using the comet assay and the micronucleus test. The cells were treated with three different concentrations (0.05, 0.1 and 0.15) of teas prepared from a 2.5% aqueous solution, under three different temperatures: (1) room (20–25°C); (2) ice-cold (2–8°C); and (3) warm (60°C). The teas were applied in co-, pre- and post-treatments in combination with the mutagen methyl methanesulfonate (MMS; 1.6×10 −4 and 4×10 −4 M). The duration of the treatment was 1 h in the comet assay and 2 h in the micronucleus test. The results showed that the mushroom was not mutagenic itself. Nevertheless, the mushroom is an efficient antimutagen against the induction of micronuclei by MMS in all concentrations and preparations tested. The observed reductions in the frequencies of micronuclei ranged from 61.5 (room temperature 0.1% tea in post-treatment) to 110.3% (co-treatment with warm and ice-cold 0.15% tea). In the comet assay, the antimutagenic activity was detected only when the cells were pre-treated with the following teas: warm 0.1 and 0.15%, room temperature 0.05% and ice-cold 0.1%. The results indicate that the mushroom A. blazei extracts are antimutagenic when tested in V79 cells.

Evaluation of a tissue homogenization technique that isolates nuclei for the in vivo single cell gel electrophoresis (comet) assay: a collaborative study by five laboratories

We evaluated a tissue homogenization technique that isolates nuclei for use in the in vivo comet assay. Five laboratories independently tested the technique using the liver, kidney, lung, spleen, and bone marrow of untreated and mutagen-treated male CD-1 mice. The direct mutagen methylmethanesulfonate (MMS) or the promutagen diethylnitrosamine (DEN) were injected intraperitoneally at maximum tolerated doses. Three and twenty-four hours later, the organs were removed and, except for bone marrow, were minced and homogenized and a nuclear suspension was prepared. The nuclear suspensions and bone marrow cells were used in the comet assay. None of the nuclear suspensions from the non-treated mice induced a positive response. All nuclear suspensions derived from the MMS-treated mice and those of the liver, kidney, and lung from DEN-treated mice induced positive responses in all the laboratories similarly. Reproducibility was demonstrated by five replicate studies in one laboratory. Furthermore, the organ-specific responses to MMS and DEN reflected the characteristic genotoxicity of the chemicals. We concluded from these results that the homogenization technique is a valid one to be used for mouse organs in the in vivo comet assay.

Cassia Senna Inhibits Mutagenic Activities of Benzo[a]‐pyrene, Aflatoxin B1, Shamma and Methyl Methanesulfonate

Ethanol extract of Senokot tablets (Cassia senna concentrate used as vegetable laxative), was found to be non-mutagenic while it inhibited the mutagenicity of benzo[a]pyrene, shamma, aflatoxin B1 and methyl methanesulfonate in the Ames histidine reversion assay using the Salmonella typhimurium tester strain TA98. While the Senokot extract completely inhibited the mutagenicity of promutagens (i.e. metabolic activation dependent) like benzo[a]pyrene and shamma, it reduced the mutagenic activity of the direct acting mutagen methyl methanesulfonate by only 58%. The mutagen aflatoxin B1 showed a 25-fold increase in the number of histidine revertants per plate at low concentrations (1.0-4.0 micrograms/plate) in the presence of metabolic activation system while at high concentrations (10.0-30.0 micrograms/plate) it proved to be weakly mutagenic (with a 5-fold increase in the number of histidine revertants/plate) without metabolic activation. The Senokot extract completely inhibited the mutagenic effect of low concentrations of aflatoxin B1 in the presence of metabolic activation but not that resulting from higher concentrations without metabolic activation. The results obtained with benzo[a]pyrene, shamma and aflatoxin B1 indicated that the antimutagenic effects of Senokot extract could be largely due to an interaction with the metabolic process involved in the activation of procarcinogens. However, the results obtained with methyl methanesulfonate suggested that factors in Senokot may also interact with direct mutagens to produce some antimutagenic effects. An ethanol extract of crude senna leaves found to be weakly mutagenic also inhibited (though less than Senokot) the mutagenic effect of benzo[a]pyrene suggesting that the antimutagenic principle is present in the complex plant material itself.

Cloning and sequencing of CATR1.3, a human gene associated with tumorigenic conversion.

The human squamous cell carcinoma cell line SCC83-01-82 (SCC) contains mutations in both the H-ras and p53 genes, but it exhibits a nontumorigenic phenotype in nude mice. This cell line can be converted into a cell line with a tumorigenic phenotype, SCC83-01-82CA (CA), by treatment with the mutagen methyl methanesulfonate (MMS). This indicates that additional genetic events leading to expression of a cooperating tumor susceptibility gene(s) may be required for tumorigenicity. To identify the cooperating gene(s), an expression cDNA library was made from tumorigenic Ca cells. The library DNA was transfected into nontumorigenic SCC cells and the transfected SCC cells were then injected into nude mice for the selection of a tumorigenic phenotype. Tumors developed in 3 of the 18 mice after injection. Several new cell lines were established from these transfected cell-induced tumors and designated as CATR cells. Tumor histology and karyotype analysis of these cells indicated that they were of human epithelial cell origin. All the CATR cells have the library vector sequence integrated in their genome. Cell line CATR1 expressed a single message from the integrated library representing a 1.3-kb cDNA insert that was absent from untransfected SCC cells or MMS-converted CA cells. This 1.3-kb cDNA insert was cloned by PCR amplification of reverse-transcribed CATR1 total RNA and was designated CATR1.3. The nucleotide sequence of CATR1.3 encodes a peptide of 79 amino acids, has a long 3' untranslated region, and represents an unknown gene product that was associated with the tumorigenic conversion due to the transfected expression library.