Methylation of MMP2, TIMP2, MMP9 and TIMP1 in abdominal aortic aneurysm.

OBJECTIVES: Abdominal aortic aneurysm (AAA) and its complications are among the most serious cardiovascular diseases and its occurrence has risen sharply in recent years. The aim of this pilot study is to explore the relationship between the methylation of matrix metalloproteinases and tissue inhibitors of the metalloproteinases genes' promoter region, and abdominal aortic aneurysm (AAA) through the detection of the methylation status of MMP2, TIMP2, TIMP1, and MMP9 genes in peripheral blood. METHODS: The study included 43 males with verified AAA (case group) and 34 healthy males (control group). The methylation status of the genes' promoter region was detected by methylation-specific polymerase chain reaction (MS-PCR). RESULTS: In adominal aortic aneurysm patients, the methylation ratio of MMP2 gene was positive in 9.3 % (4 cases), 2.3 % (1 case) had methylated TIMP2 gene, 7.0 % (3 cases) had methylated TIMP1 gene, while the methylation ratio of MMP9 gene was positive in 93.0 % (40 cases). In the control group, MMP2 gene was found to be methylated in 5.9 % (2 cases), 5.9 % of cases had methylated TIMP2 and TIMP1 genes (2 cases), and MMP9 gene was found to be methylated in 91.2 % (31 cases). CONCLUSION: In our pilot study, we found no association between DNA methylation of gelatinases and their tissue inhibitors, and the development of an abdominal aortic aneurysm (Tab. 2, Fig. 1, Ref. 27).

[Ipsilateral Fractures of the Coracoid and Acromion Process of the Scapula Combined with the Distal Clavicle End Fracture, Treatment Options]. / Ipsilaterální zlomenina processus coracoideus a akromionu lopatky v kombinaci se zlomeninou laterální cásti klavikuly, terapeutická moznost resení.

Superior Shoulder Suspensory Complex (SSSC) is a bone and soft-tissue ring securing the connection of the upper extremity to the axial skeleton via the clavicle and sternoclavicular joint. An isolated injury to one component of SSSC is usually stable. An injury to 2 of its components is a potential source of shoulder girdle instability and requires surgical stabilisation. An injury affecting 3 and more components is extremely rare and surgical stabilisation should be indicated. Our study presents the case of a 50-year-old man who fell off the bicycle and sustained a direct blow to his left shoulder resulting in an ipsilateral fracture of the coracoid and acromion process combined with the fracture of the distal end of the clavicle. Following a standard clinical examination and a subsequent X-ray and a CT scan with three-dimensional shoulder reconstruction, an open reduction and stabilisation of all the injured SSSC components was performed. Later, early and gradual rehabilitation of the shoulder girdle was commenced. At 48 weeks after the surgery, almost full range of motion of the shoulder joint was achieved and the muscle strength of the operated upper extremity was comparable to that of the healthy one. Key words:Superior Shoulder Suspensory Complex, fracture, acromion, coracoid process, clavicle.

Predictors of the immune response to booster immunisation against tetanus in Czech healthy adults.

An evaluation of the relationship between predictors and immune response was conducted using data obtained from a clinical trial in 200 Czech healthy adults aged 24-65 years receiving a booster dose of a monovalent tetanus vaccine in 2017. The response was determined from ELISA antibody concentrations of paired sera obtained before and 4 weeks after the immunisation. While all subjects with initial antibody levels 2.2 IU/ml. The immune response was not affected by sex, age, tetanus vaccine type, concomitant medication, related adverse events or post-vaccination period since there were no significant differences in geometric mean concentrations or seroconversion rates. The seroconversion rate of 56% in smokers was significantly lower than that of 73% achieved in non-smokers. Although the seroconversion rates did not differ between individuals with normal or higher body weight, the adjusted odds ratio (1.3; 95% Cl 1.08-1.60) revealed a positive correlation between seroconversion rate and body mass index (BMI). Although the vaccine-induced response was influenced by pre-vaccination antibody levels, smoking or BMI, the booster immunisation against tetanus produced a sufficient response regardless the predictors.

Association between interleukin-18 variants and prostate cancer in Slovak population.

Interleukin-18 (IL-18), pro-inflammatory cytokine, plays important role in antitumor immunity. Polymorphisms in the IL-18 gene may lead to its altered production/activity and such modulate susceptibility to prostate cancer. The aim of this study was to evaluate the relationship between the -607 and +105 polymorphisms in the IL-18 gene and the risk of prostate cancer development and progression in Slovak population. The study was performed using 425 patients with prostate cancer, 270 patients with benign prostatic hyperplasia (BHP) and 263 healthy male controls. The statistically significant association of the -607 AC genotype (OR = 2.24; p < 0.001), CC genotype (OR = 1.86; p = 0.006), as well as C allele (OR = 1.27; p = 0.033) with the higher risk of prostate cancer development was observed. No association of the IL-18 -607 polymorphism and BHP was detected. The subset analysis revealed the significant association of the -607 AC genotype (OR = 2.01; p = 0.008) with development of higher-grade carcinomas (Gleason score ≥7) and the strong association of the -607 AC genotype (OR = 3.11; p < 0.001), CC genotype (OR = 2.96; p < 0.001) as well as C allele (OR = 1.51; p = 0.003) with the higher risk of prostate cancer development in the group of patients with PSA < 10 ng/ml. The -607 AC genotype was also connected with significantly higher IL-18 plasma concentrations. No association between the IL-18 +105 polymorphism and prostate cancer was observed. The analysis of the distribution of the -607 and +105 haplotypes showed significant association of the - 607 C/ + 105 A and - 607 C/ + 105 C haplotypes with the risk of prostate cancer. This study found that the IL-18 -607 promoter polymorphism could contribute to prostate cancer development in Slovak population. Its presence was also associated with development of higher-grade carcinomas and therefore may influences the prognosis and aggressiveness of the disease.

N-acetylcysteine alleviates the meconium-induced acute lung injury.

Meconium aspiration in newborns causes lung inflammation and injury, which may lead to meconium aspiration syndrome (MAS). In this study, the effect of the antioxidant N-acetylcysteine on respiratory and inflammatory parameters were studied in a model of MAS. Oxygen-ventilated rabbits were intratracheally given 4 mL/kg of meconium (25 mg/mL) or saline. Thirty minutes later, meconium-instilled animals were administered N-acetylcysteine (10 mg/kg; i.v.), or were left without treatment. The animals were oxygen-ventilated for additional 5 h. Ventilatory pressures, oxygenation, right-to-left pulmonary shunts, and leukocyte count were measured. At the end of experiment, trachea and lung were excised. The left lung was saline-lavaged and a total and differential count of cells in bronchoalveolar lavage fluid (BAL) was determined. Right lung tissue strips were used for detection of lung edema (expressed as wet/dry weight ratio) and peroxidation (expressed by thiobarbituric acid-reactive substances, TBARS). In lung and tracheal strips, airway reactivity to acetylcholine was measured. In addition, TBARS and total antioxidant status were determined in the plasma. Meconium instillation induced polymorphonuclear-derived inflammation and oxidative stress. N-acetylcysteine improved oxygenation, reduced lung edema, decreased polymorphonuclears in BAL fluid, and diminished peroxidation and meconium-induced airway hyperreactivity compared with untreated animals. In conclusion, N-acetylcysteine effectively improved lung functions in an animal model of MAS.

Why mitochondria are excellent targets for cancer therapy.

New insights into cancer cells - specific biological pathways are urgently needed to promote development of exactly targeted therapeutics. The role of oncoproteins and tumor suppressor proteins in proliferative signaling, cell cycle regulation and altered adhesion is well established. Chemicals, viruses and radiation are also generally accepted as agents that commonly induce mutations in genes encoding these cancer-inducing proteins, thereby giving rise to cancer. More recent evidence indicates the importance of two additional key factors imposed on proliferating cells - hypoxia and/or lack of glucose. These two additional triggers can initiate and promote the process of malignant transformation, when a low percentage of cells escape cellular senescence. Disregulated cell proliferation leads to formation of cellular masses that extend beyond the resting vasculature, resulting in oxygen and nutrient deprivation. Resulting hypoxia triggers a number of critical adaptations that enable cancer cell survival. The process of apoptosis is suppressed and glucose metabolism is altered. Recent investigations suggest that oxygen depletion stimulates mitochondria to compensate increased reactive oxygen species (ROS). It activates signaling pathways, such as hypoxia-inducible factor 1, that promote cancer cell survival and tumor growth. During the last decade, mitochondria have become key organelles involved in chemotherapy-induced apoptosis. Therefore, the relationship between mitochondria, ROS signaling and activation of survival pathways under hypoxic conditions has been the subject of increased study. Insights into mechanisms involved in ROS signaling may offer novel ways to facilitate discovery of cancer-specific therapies.

NMR plasma metabolomics study of patients overcoming acute myocardial infarction: in the first 12 h after onset of chest pain with statistical discrimination towards metabolomic biomarkers.

Acute myocardial infarction (AMI) is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia is essential for the ischemic patient's prevention and treatment. Despite the great prevalence and incidence only a small number of studies utilize a metabolomic approach to describe AMI condition. Recent studies have shown the impact of metabolites on epigenetic changes, in these studies plasma metabolites were related to neurological outcome of the patients making metabolomic studies increasingly interesting. The aim of this study was to describe metabolomic response of an organism to ischemic stress through the changes in energetic metabolites and aminoacids in blood plasma in patients overcoming acute myocardial infarction. Blood plasma from patients in the first 12 h after onset of chest pain was collected and compared with volunteers without any history of ischemic diseases via NMR spectroscopy. Lowered plasma levels of pyruvate, alanine, glutamine and neurotransmitter precursors tyrosine and tryptophan were found. Further, we observed increased plasma levels of 3-hydroxybutyrate and acetoacetate in balance with decreased level of lipoproteins fraction, suggesting the ongoing ketonic state of an organism. Discriminatory analysis showed very promising performance where compounds: lipoproteins, alanine, pyruvate, glutamine, tryptophan and 3-hydroxybutyrate were of the highest discriminatory power with feasibility of successful statistical discrimination.

Double-blind, placebo-controlled evaluation of grass pollen specific immunotherapy with oral drops administered sublingually or supralingually.

Forty-one patients suffering from grass pollen allergy underwent specific immunotherapy with standardized allergen extract consisting of six grass pollens (H-Al per os) administered either sublingually or supralingually for one year. In order to investigate clinical and immunological changes induced by the administration of allergens via the oral mucosa, the double-blind, placebo-controlled, randomized design of the trial with 30 other patients enrolled in placebo groups was applied. Specific immunotherapy with oral drops administered sublingually or supralingually was performed in the same way, keeping the drops under or on the tongue, respectively, for 1-2 min before swallowing them; at the end of the trial the cumulative dose of the allergen was almost 20 times higher than that of the subcutaneous therapy with corresponding allergen preparation. Data about symptoms scores and drugs intake during grass pollen season, as well as skin reactivity, levels of specific IgG and IgE antibodies, before the study and after the study's completion, were obtained. It was found that both routes of administration are effective according to subjective clinical parameters and drug consumption, with a highly significant reduction of symptoms and drug intake favoring sublingual administration where a reduction of more than 60% was achieved. Only sublingual active group showed a significant increase in Dactylis glomerata-specific IgG serum levels. Adverse effects were limited to a small number of generally mild local and/or systemic reactions. The results suggest that the administration of allergens via the oral mucosa is safe and clinically effective, favoring the sublingual rather than supralingual route.

Human adipose tissue accumulation is associated with pro-inflammatory changes in subcutaneous rather than visceral adipose tissue.

The importance of the involvement of adipose tissue macrophage subpopulations in obesity-related disorders is well known from different animal models, but human data are scarcer. Subcutaneous (n=44) and visceral (n=52) adipose tissues of healthy living kidney donors were obtained during living donor nephrectomy. Stromal vascular fractions were isolated and analysed by flow cytometry using CD14, CD16, CD36 and CD163 antibodies. Total macrophage numbers in subcutaneous adipose tissue increased (P=0.02) with body mass index (BMI), with a similar increase seen in the proportion of phagocytic CD14+CD16+CD36high macrophages (P<0.01). On the other hand, there was an inverse correlation between anti-inflammatory CD14+CD16-CD163+ macrophages (P<0.05) and BMI. These correlations disappeared after excluding obese subjects (BMI ⩾30 kg m-2) from the analysis. Interestingly, none of these subpopulations were significantly related to BMI in visceral adipose tissue. Obesity per se is associated with distinct, highly phagocytic macrophage accumulation in human subcutaneous adipose tissue.

Isolation and properties of serum albumin from the house mouse Mus musculus.

Precipitation of serum proteins of the house mouse, Mus musculus, by trichloroacetic acid and redissolving part of the precipitate in ethanol proved to be a very efficient procedure for isolating mouse serum albumin. Electrophoretic and immunoelectrophoretic analyses showed only albumin to be dissolved at a detectable level in the ethanol. Electrophoretic studies also showed that isolated albumin underwent polymerization with the polymers being readily separated by exculsion chromatography on Sephadex G-150. Comparisons of such physicochemical characteristics of the albumin monomers, as extinction coefficient (5.2), sedimentation coefficient (4.5 S), molecular weight (67 000), free sulfhydral groups per molecule (0.6) and amino acid composition with albumins of other mammals showed strong similarity. Finally, a comparison was made between the common mouse albumin or albumin A, and a variant, albumin C. The two types of albumin molecules were found very similar in all respcets except electrophoretic migration.

Evolutionary relationships among five subspecies of Mus musculus based on restriction enzyme cleavage patterns of mitochondrial DNA.

The intra- and intersubspecific genetic distances between five subspecies of Mus musculus were estimated from restriction enzyme cleavage patterns of maps of mitochondrial DNA (mtDNA). The European subspecies, M. m. domesticus and Asian subspecies, M. m. bactrianus, M. m. castaneus, M. m. molossinus and M. m. urbanus were examined. For each subspecies, except M. m. urbanus, at least two local races from widely separated localities were examined. Intrasubspecific heterogeneity was found in the mtDNA cleavage patterns of M. m. bactrianus and M. m. castaneus. M. m. molossinus and M. m. domesticus, however, revealed no intrasubspecific heterogeneity. Four of the subspecies had distinct cleavage patterns. The fifth, M. m. urbanus, had cleavage patterns identical to those of M. m. castaneus with several enzymes. Estimates of genetic distances between the various races and subspecies were obtained by comparing cleavage maps of the mtDNAs with various restriction enzymes. Nucleotide sequence divergences of mtDNA between local races were estimated to be less than 0.4% in M. m. bactrianus and less than 0.3% in M. m. castaneus. The times of divergence of both subspecies were calculated to be 0.1--0.2 x 10(6) years. These values suggest that the intrasubspecific divergence began some 0.1--0.2 x 10(6) years ago. On the other hand, nucleotide sequence divergences between European subspecies M. m. domesticus and Asian subspecies M. m. bactrianus and M. m. castaneus were 7.1% ane 5.8%, respectively. The times of divergence were calculated to be 2.1--2.6 x 10(6) years. Further, the nucleotide sequence divergence and time of divergence between M. m. molossinus and the other two Asian subspecies were comparable to those between M. m. molossinus and M. m. domesticus (about 3% and 1 x 10(6) years, respectively). These results suggest that M. m. molossinus is situated in a unique evolutionary position among Asian subspecies.

Comparison of three power supplies used for the single-cell gel assay.

The alkaline single-cell gel (SCG) assay, which determines DNA damage in mice treated with 100 mg/kg methyl methanesulfonate (MMS), was run by using three power supplies from Bio-Rad (models 3000Xi, 200/2.0, and Power Pac 300). Comparisons of the results obtained from the use of these power supplies showed differences in mean DNA tail length to width ratios and profiles of these ratios in individual cells. Model 200/2.0 power supply appeared to enhance significantly the sensitivity of the assay. In purchasing power supplies, there is a need to evaluate their effect on the sensitivity of the test.

Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay.

To monitor genotoxicity in small bodies of water (e.g., creeks, ponds, and drainage ditches) we examined tadpole erythrocytes of two species: Rana clamitans and Rana pipiens,using the alkaline single cell gel DNA electrophoresis (SCG) or "comet" assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a "comet with tail" formation. Fifty-six samples, a total of 606 tadpoles, from 18 sites in southern Ontario, collected between 1993 and 1995, were examined. Samples of R. clamitans tadpoles collected in 1994 and 1995, from regions with heavy agricultural activity, gave significantly higher (P < 0.001) DNA length to width ratios than samples of R. clamitans tadpoles collected from sites in the Bruce Peninsula and near the French River, which have little or no agriculture. Samples of R. pipiens tadpoles collected in 1994 from sites on the outskirts of Windsor, Ontario, sites which receive genotoxic inputs from nearby industries, gave significantly higher (P < 0.001) DNA ratios than samples from agricultural areas and the Bruce Peninsula. R. clamitans tadpoles showed significant annual variation in DNA damage which was greater in samples of tadpoles collected from agricultural areas than from the Bruce Peninsula. The higher levels of DNA damage in tadpoles collected from agricultural areas may be due to the pesticides used, and the increased variation in DNA damage in the same areas is likely due to the impact of crop rotation, including leaving fields fallow, the timing of rainfall, and/or the application of pesticides. R. clamitans tadpoles, especially those collected from agricultural areas, also showed significant seasonal variation in DNA damage. There was no significant (P > 0.05) seasonal or annual variation in the levels of DNA damage in R. pipiens tadpoles collected from the Tallgrass Prairie. This study indicates that both species are suitable for use in the alkaline SCG assay and as in situ sentinel organisms for environmental biomonitoring.

Comparison of sensitivity to methyl methanesulphonate among tadpole developmental stages using the alkaline single-cell gel electrophoresis (comet) assay.

In a previous study, we demonstrated that tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) for genotoxicity using the alkaline single-cell gel DNA electrophoresis (SCG) or "comet" assay [Ralph and Petras, 1997]. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a "comet with tail" formation. In this initial study, most of the tadpoles collected were in the early stages of larval development, but this is not always possible. The present study evaluated the sensitivity of tadpoles, at different stages of larval development, to a range of concentrations of the genotoxicant methyl methane-sulphonate (MMS). Four specific phases of Rana clamitans (green frog) larval development were examined: first-year limbless tadpoles (Stage I as defined by Taylor and Kollros [1946]), second-year limbless tadpoles (Stages II-III), second-year tadpoles with only hindlimbs (Stages X-XVIII), and second-year tadpoles with all four limbs evident and a tail undergoing resorption (Stages XXII-XXIII). Twenty-four hour exposures to MMS of tadpoles in the three earliest phases produced a significant (P < 0.01) added variance component among tadpoles for DNA damage and there were significant increases (P < 0.05) in the length:width ratios of the DNA patterns at concentrations as low as 1.56 mg/I. However, tadpoles in the last phase studied (both pairs of limbs present) showed no significant (P > 0.05) added variance component and no significant increases (P> 0.05) in DNA damage upon exposure to any of the MMS doses tested. A nested ANOVA indicated that, for each of the tested concentrations of MMS, but not the dechlorinated water control, there was significant heterogeneity (P < 0.05) in DNA damage when tadpoles of all four phases studied were compared. However, when tadpoles of the lost phase of development were removed from the comparison, there was no significant heterogeneity (P > 0.05) among tadpoles of the remaining three phases. Possible reasons for this insensitivity to MMS as animals enter the metamorphic climax were considered. The results indicate that pooling of the early tadpole phases of R. clamitans for SCG environmental genotoxicity biomonitoring is acceptable.

Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (comet) assay.

Pesticides are broadly used for pest control in agriculture despite possible negative impacts they may pose to the environment. Thus, we examined the DNA damage caused by five herbicides commonly used in southern Ontario (Canada). Erythrocytes from Rana catesbeiana (bullfrog) tadpoles were evaluated for DNA damage following exposure to selected herbicides, using the alkaline single-cell gel DNA electrophoresis (SCG) or "comet" assay [Singh et al. (1988): Exp Cell Res 175:184-191; Ralph et al. (1996): Eviron Mol Mutagen 28:112-120]. This approach involves detection, under alkaline conditions, of DNA fragments that upon electrophoresis migrate from the nuclear care, resulting in a comet formation. The herbicides tested, along with their active ingredients, were AAtrex Nine-O (atrazine), Dual-960E (metalochlor), Roundup (glyphosate), Sencor-500F (metribuzin), and Amsol (2,4-D amine). Tadpoles were exposed in the laboratory for a 24-hr period to several concentrations of the herbicides dissolved in dechlorinated water. Methyl methanesulphonate was used as a positive control. The herbicides AAtrex Nine-O-, Dual-960E-, Roundup-, and Sencor-500F-treated tadpoles showed significant DNA damage when compared with unexposed control animals, whereas, Amsol-treated tadpoles did not. Unlike the other responding herbicides, Sencor-500F did not show a relationship between dosage and DNA damage. In summary, the results indicate that at least some of the herbicides currently used in southern Ontario are capable of inducing DNA damage in tadpoles.

Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles.

Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) ("comet") assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a "comet-with-tail" formation. Exposure of R. clamitans todpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans todpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment.

Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp.

Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (SCG) or "comet" assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which, on electrophoresis, migrate from the nuclear core, resulting in a "comet with tail" formation. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery-bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants.

Comparison of DNA damage in peripheral blood and spleen lymphocytes using the single-cell gel assay.

The alkaline single-cell gel (SCG) or 'comet' assay has been applied to the detection of DNA damage from a number of chemical and biological factors in vivo and in vitro. In the past, a number of cell types has been used with peripheral blood lymphocytes being the most readily accessible. This study was designed to determine whether lymphocytes sequestered in the spleen might prove more sensitive to DNA damage than those in the peripheral circulation. This would result in a more effective SCG assay. Baseline DNA length to width ratios for peripheral blood and splenic lymphocytes did not differ significantly from each other (1.27 and 1.21, respectively). Neither did ratios of lymphocytes from the two sources, sampled 20 and 48 h after injection with 100 mg/kg methyl methanesulfonate (MMS) (3.81 and 3.62 at 20 h, respectively; and 1.96 and 2.21 at 48 h, respectively). Recovery from MMS damage at 168 h postinjection was also not different in the two groups of cells (1.13 and 1.16, respectively). However, an examination of cell profiles of DNA damage showed that splenic lymphocytes had a significantly higher percentage of damaged cells (63.33%) than did peripheral blood lymphocytes (40.67%) 48 h postinjection. Of the hypotheses proposed for this difference, the most likely seems to involve the different proportions of B- and T-lymphocytes present in the peripheral blood and the spleen. Since the difference between peripheral blood and splenic lymphocytes was seen only at 48 h postinjection, the use of splenic lymphocytes in the SCG assay is not advantageous under most circumstances.

Caged amphibian tadpoles and in situ genotoxicity monitoring of aquatic environments with the alkaline single cell gel electrophoresis (comet) assay.

In previous studies we demonstrated that indigenous amphibian tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) using the alkaline single cell gel electrophoresis (SCG) or 'comet' assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a 'comet with tail' formation. However, although often plentiful, tadpoles are not present in all aquatic environments. Both larger bodies of water (e.g., lakes and rivers) and those impacted upon heavily by man (e.g., bodies of water near industrial sites, on landfills, and in urban areas) often do not support amphibian tadpole populations. An alternative approach to the collection of indigenous tadpoles is to place caged tadpoles at these sites for short term exposures to environmental contaminants. To determine the feasibility of such an approach, Rana clamitans (green frog) and Bufo americanus (American toad) tadpoles were housed in cages at 11 sites in southwestern Ontario (Canada). In a preliminary experiment, we found that tadpoles caged at a polluted reference site (Tallgrass Prairie Heritage Park in Windsor, Ontario) for either 7 or 14 days showed significant (P < 0.05) increases in DNA damage, relative to tadpoles caged in the laboratory in dechlorinated water. As a result we routinely used a 7 day exposure time. Significantly (P < 0.05) increased levels of DNA damage, relative to their controls, were observed in tadpoles caged at three sites along two creeks draining a large petrochemical installation south of Sarnia, Ontario; at two sites in the Tallgrass Prairie Heritage Park; and at a site along the Ecarte Channel which is part of the St. Clair River. The DNA damage levels of animals caged in Lake St. Clair, in the Trenton Channel of the Detroit River, at a landfill site, and in two creeks in the city of Windsor did not differ significantly (P > 0.05) from their controls. This study demonstrates that caged tadpoles are suitable for monitoring most bodies of fresh water, particularly those aquatic habitats mentioned above where indigenous tadpoles are not present. A combined approach of collecting indigenous tadpoles and using caged tadpoles should provide a sensitive system for aquatic genotoxicity monitoring.

Comparison of alkaline single cell gel (Comet) and peripheral blood micronucleus assays in detecting DNA damage caused by direct and indirect acting mutagens.

The alkaline single cell gel (SCG) or 'comet' and peripheral blood micronucleus (pbMN) assay have been used to compare the effects of the direct acting mutagens, methyl methanesulfonate (MMS) and N-nitroso-N-methylurea (NMU), and the indirect acting mutagens, benzo[a]pyrene (BAP), cyclophosphamide (CP) 9, 10-dimethyl-1,2-benzanthracene (DMBA), and mitomycin C (MMC) in an inbred house mouse (Mus domesticus) strain. The alkaline SCG assay was able to detect DNA damage from direct acting mutagens. However, it appears that, even at the highest concentrations tested, the SCG assay was not able to detect DNA damage caused by 3 of 4 indirect acting mutagens tested. The exception was BAP. The pbMN assay was sensitive to DNA damage caused by both groups of mutagens. Multiple injections did not increase the sensitivity of the SCG assay to the indirect acting mutagen CP. Further, simultaneous injections of CP and MMS, in one experiment, resulted in significantly lower (p < 0.05) average DNA ratios and micronucleated polychromatic erythrocyte counts than those obtained after treatment with MMS alone. Although the SCG assay has been shown to be sufficiently sensitive to detect DNA damage caused by both direct and indirect acting mutagens in deermice (Peromyscus maniculatus) and bullheads (Ameiurus nebulosus), similar results are not seen in the inbred house mouse strain tested.