Induction of sister-chromatid exchanges by mutagens from amino acid and protein pyrolysates

Sister-chromatid exchanges (SCEs) in a permanent cell line of human lymphoblastoid cells were induced by 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]-indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyridol[1,2-a:3′,2′-d]imidazole (Glu-P-1) and 2-amino-9H-pyrido[2,3-b]indole (2-amino-α-carboline). The first two compounds were found in tryptophan pyrolysates, the third in a glutamic acid pyrolysate and the last in a globulin pyrolysate. All these compounds required the metabolic activation system (S9 mix) for induction of SCE. Trp-P-2 had the highest SCE-inducing activity of these chemicals (approximately equivalent to that of aflatoxin B₁), followed by Trp-P-1, Glu-P-1 and then 2-amino-α-carboline.     

Degradation of mutagens from pyrolysates of tryptophan, glutamic acid and globulin by myeloperoxidase.

Mutagenic compounds isolated from pyrolysates of tryptophan, glutamic acid and globulin were broken down by myeloperoxidase and hydrogen peroxide with loss of their mutagenicity toward Salmonella typhimurium TA98. Lactoperoxidase and horseradish peroxidase were as effective as myeloperoxidase in degradation of the mutagens.     

Environmental mutagens may be implicated in the emergence of drug-resistant microorganisms

The emergence of drug-resistant microorganisms is an important medical and social problem. Drug-resistant microorganisms are thought to grow selectively in the presence of antibiotics. Most clinically isolated drug-resistant microorganisms have mutations in the target genes for the drugs. While any of the many mutagens in the environment may cause such genetic mutations, no reports have yet described whether these mutagens can confer drug resistance to clinically important microorganisms. We investigated how environmental mutagens might be implicated in acquired resistance to antibiotics in clinically important microorganisms, which causes human diseases. We selected mutagens found in the environment, in cigarette smoke, or in drugs, and then exposed Pseudomonas aeruginosa to them. After exposure, the incidence of rifampicin- and ciprofloxacin-resistant P. aeruginosa strains markedly increased, and we found mutations in genes for the antibiotic-target molecule. These mutations were similar to those found in drug-resistant microorganisms isolated from clinical samples. Our findings show that environmental mutagens, and an anticancer drug, are capable of inducing drug-resistant P. aeruginosa similar to strains found in clinical settings.     

Molecular identification of mutations in G6PD gene in patients with favism in Iran

Glucose 6-phosphate dehydrogenase is a highly polymorphic enzyme encoded by a human X-linked gene (Xq2.8). This enzyme catalyses the first step of pentose phosphate pathway, that converts glucose 6-phosphate to 6-phosphogluconate with production of NADPH2. G6PD deficiency is the most common human metabolic inborn error affecting more than 400 million people world wide. The main clinical manifestations are acute hemolytic anemia and jaundice, triggered by infection or ingestion of Fava beans or oxidative drugs. A predominant variant of G6PD named Mediterranean is often associated with favism. This has been evident in several countries including Northern coastal provinces of Iran. Other current variants are Chatham and Cosenza. Molecular identification of the most prevalent mutations in G6PD gene was carried out in 71 males and females with G6PD deficiency. They were from Iranian Northern province of Golestan. DNA was extracted from blood samples and analyzed for known G6PD mutation by PCR and restriction fragment length polymorphisms (RFLP) technique. Adapting this method, revealed that Mediterranean mutation at nt 563(C-->T) is predominant in the area (69%) and 26.7% of patients have Chatham mutation at nt 1003(G-->A). Findings indicate a higher prevalence of these mutations, in Golestan compared to Mazandaran (66.2% Mediterranean and 19% Chatham mutation) and Gilan (86.4% Mediterranean and 9.71% Chatham mutations). Cosenza mutation at nt 1376(G-->C), by PCR-RFLP technique was not found among other 3 samples (4.3%). The similarity of these results with mutations in Italy indicates probable existence of a common ancestral origin in the observed populations.     

Endogenous mutagens and the causes of aging and cancer

A very large oxidative damage rate to DNA occurs as part of normal metabolism. In each rat cell the steady-state level is estimated to be about 10⁶ oxidative adducts and about 10⁵ new adducts are formed daily. It is argued that this endogenous DNA damage is a major contributor to aging and the degenerative diseases of aging, such as cancer. The oxidative damage rate in mammalian species with a high metabolic rate, short life span, and high age-specific cancer rate is much higher than the rate in humans, a long-lived creature with a lower metabolic rate and a lower age-specific cancer rate. It is argured that deficiency of micronutrients, such as dietary antioxidants or folate, is a major contributor to human cancer and degenerative diseases.

Understanding the role of mitogenesis in mutagenesis is critical for clarifying the mechanisms of carcinogenesis and interpreting high-dose animal cancer tests. High-dose animal cancer tests have been done mainly on synthetic industrial chemicals, yet almost all of the chemicals humans are exposed to are natural. About half of natural chemicals tested in high-dose animal cancer tests are rodent carcinogens, a finding that is consistent with the view that high-dose tests frequently increase mitogenesis rates. Animals have numerous defenses against toxins that make them very well buffered against low doses of almost all toxins, whether synthetic or natural.     

Characterization of aminoalkylimidazol-4-one mutagens from liquid-reflux models

A new class of low molecular weight, aminomethylimidazol-4-one (IQ-"like") mutagens have been produced by the reaction of creatinine with the amino acid L-threonine, in liquid-reflux models, mimicking cooking, of diethylene glycol:5% distilled water (2 h at 150 degrees C). Two mutagens, 2-amino-1-methyl-5-propylideneimidazol-4-one (AMPI) and 2-amino-5-ethylidene-1-methylimidazol-4-one (AEMI) were isolated and characterized by UV absorption spectra, mass spectra, and 1H-NMR. The mutagen AEMI was identical to that obtained from the reaction of creatinine with acetaldehyde. These mutagens were positive in all IQ-sensitive Ames tester strains and were not inactivated by acidic nitrosation at pH 1.0. Products displaying mutagenicity were also obtained by refluxing creatinine with other hydroxyamino acids such as L-serine, L-homoserine, and L-4-amino-3-hydroxybutyric acid, and aldehydes such as glyoxal, methylglyoxal, glycolaldehyde, but not formaldehyde. Simple model systems such as creatinine and acetaldehyde may be useful in more clearly defining the exact mechanism of formation of IQ-type mutagens (aminomethylimidazo-quinolines and -quinoxalines) produced during cooking, as well as in screening for potential inhibitors of IQ-type mutagen formation, and elucidating the mechanism of such inhibition.     

Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.     

Characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis

Human angiogenin (ANG), the first member of the angiogenin family (from the pancreatic ribonuclease A superfamily) to be identified, is an angiogenic factor that induces neovascularization. It has received much attention due to its involvement in the growth of tumors and its elevated expression level in pancreatic and several other cancers. Recently the biological role of ANG has been shown to extend to the nervous system. Mutations in ANG have been linked with familial as well as sporadic forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by selective destruction of motor neurons. Furthermore, mouse angiogenin-1 has been shown to be expressed in the developing nervous system and during the neuronal differentiation of pluripotent stem cells. We have now characterized the seven variants of ANG reported in ALS patients with respect to the known biochemical properties of ANG and further studied the biological properties of three of these variants. Our results show that the ribonucleolytic activity of six of the seven ANG-ALS implicated variants is significantly reduced or lost and some variants also show altered thermal stability. We report a significant reduction in the cell proliferative and angiogenic activities of the three variants that we chose to investigate further. Our studies on the biochemical and structural features of these ANG variants now form the basis for further investigations to determine their role(s) in ALS.     

Detection and identification of mutagens and carcinogens as their adducts with guanosine derivatives

For use in screening for environmental mutagens and carcinogens, a highly fluorescent derivative of guanosine, 2'-deoxy-2'-(2",3"-dihydro-2",4"-diphenyl-2"-hydroxy-3"-oxo-1"-pyrrol yl) guanosine (FG), was synthesized. When incubated with FG in aqueous solution, mutagens form adducts that can be analyzed with an HPLC-fluorescence detector-system. By this method, mutagens such as glyoxal, methylglyoxal, 2-(2-furyl)-3-(5-nitrofuryl) acrylamide and 4-nitroquinoline-N-oxide, used as model compounds, were detected rapidly with high sensitivity. Reaction with isopropylideneguanosine (IPG), followed by isolation and characterization of the mutagen-IPG-adduct was found to be a useful method for identifying unknown mutagens in crude samples. This method was successfully applied in identification of the mutagens in heated glucose (200 degrees C, 20 min); glyoxal-IPG and 8-hydroxy-IPG were identified in the reaction mixture.     

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.     

Isolation and Partial Characterization of a Tenuivirus from Wheat in Iran

A tenuivirus, tentatively designated Iranian wheat stripe virus (IWSV), was transmitted by the delphacid Unkanodes tanasijevici and induced dwarfing, striping and yellowing symptoms in wheat. It could also infect barley, oat, rice, rye, sorghum and a number of other gramineous species. The virus was purified from wheat by chloroform clarification and differential and density-gradient centrifugation. It formed several layers in density-gradient columns. Purified virus preparations had a UV absorption spectrum typical of nucleoproteins and contained flexuous supercoils of 8.7 nm and fine filaments of 4.3 nm width. The antiserum produced against the virus reacted with infected plant sap in agar-gel diffusion and enzyme-linked immunosorbent assay (ELISA). The virus could be readily detected in individual viruliferous planthoppers by ELISA. IWSV was serologically related to rice hoja blanca virus but not to maize stripe virus. The relationship of IWSV with other tenuiviruses is discussed.     

Molecular Characterization of Cryptosporidium Isolates from Humans and Animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.