Benzo[a]pyrene-enhanced mutagenesis by man-made mineral fibres in the lung of lamda-lacI transgenic rats.

In an attempt to examine the interaction of man-made mineral fibres with benzo[a]pyrene (B[a]P), homozygous X-lacI transgenic F344 rats were intratracheally treated with rock (stone) wool RWI and glass wool MMVF 10 fibres together with B[a]P. To analyze the induction of gene mutations by fibres and B[a]P in lung, single doses of 1 and 2 mg fibres/animal or multiple doses of 2 mg fibres/animal were administered weekly on 4 consecutive weeks (total dose 8 mg/animal). B[a]P (10 mg/animal) was administered either simultaneously with fibres (for single dose treatment with fibres) or together with the last fiber treatment (for multiple dose treatment with fibres). Animals were scarified 4 weeks after the last treatment. Benzo[a]pyrene administered simultaneously with RW1 fibres exhibited a strong synergistic effect on mutagenicity, the observed mutant frequency (MF) being more than three-fold higher than the net sum of the MF induced after separate administration of both agents. Our data suggest that DNA adducts induced by simultaneous B[a]P and fiber treatment lead to a strong increase in mutatant frequencies.

Mutagenesis by man-made mineral fibres in the lung of rats.

The potential of two asbestos substitute mineral fibres--rock (stone) wool RW1 and glass wool MMVF10--to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous lamda-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple doses of 2 mg/animal administered weekly on four consecutive weeks (8 mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1 mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RWl fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4 x 2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres.

Postural and trunk responses to unexpected perturbations depend on the velocity and direction of platform motion.

This study compares postural and trunk responses to translating platform perturbations of varied velocities and directions. A group of 18 young and physically active subjects were exposed to a set of postural perturbations at varied velocities (5, 10, 15, and 20 cm/s) and directions of platform movement (forward, backward, left-lateral, and right-lateral). The center of pressure (CoP) displacement measurement, in addition to the trunk motion (representing the center of mass (CoM) displacement), were both monitored. Results identified that the CoP displacement increased from slow to faster velocities of platform motion more widely in both anterior and posterior directions (50.4 % and 48.4 %) as compared to the CoM displacement (17.8 % and 14.9 %). However a greater increase in the peak CoM velocity (70.3 % and 69.6 %) and the peak CoM acceleration (60.5 % and 53.1 %) was observed. The values in the anterior and posterior direction only differed significantly at the highest velocity of platform motion (i.e. 20 cm/s). A similar tendency was observed in the medio-lateral direction, but there were no significant differences in any parameter in the left-lateral and right-lateral direction. The velocity of the platform motion highly correlated with peak velocity (r=0.92-0.97, P<0.01) and moderately with amplitude of trunk displacement (r=0.56-0.63, P<0.05). These findings indicate that the velocity of perturbation alters peak CoM velocity rather than the magnitude of CoM displacement. The effect of the direction of perturbations on the trunk response emerges only at a high velocity of platform motion, such that the peak CoM velocity and peak CoM acceleration are significantly greater in anterior than posterior direction.

Ontogenesis of photoperiodic entrainment of the molecular core clockwork in the rat suprachiasmatic nucleus.

The molecular mechanism underlying a generation of circadian rhythmicity within the suprachiasmatic nucleus (SCN) is based on interactive negative and positive feedback loops that drive the rhythmic transcription of clock genes and translation of their protein products. In adults, the molecular mechanism is affected by seasonal changes in day length, i.e., photoperiod. The photoperiod modulates phase, waveform, and amplitude of the rhythmic clock genes expression as well as the phase relationship between their profiles. To ascertain when and how the photoperiod affects the circadian core clock mechanism during ontogenesis, the rhythmic expression of clock genes, namely of Per1, Per2, Cry1 and Bmal1 was determined in 3-, 10- and 20-day-old rat pups maintained under either a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) or under a short, LD 8:16 photoperiod. The daily profiles in the level of clock genes mRNA were studied in constant darkness. The photoperiod affected the profile of Per1 and Per2 mRNA in 20- and 10- but not yet in 3-day-old pups. Expression of Cry1 was affected only in 20-day-old pups, whereas expression of Bmal1 was not yet affected even in 20-day-old rats. The results demonstrate no effect of the photoperiod on 3-day-old pups, only partial entrainment of the molecular core clockwork in 10-day-old pups and a more mature, though not yet fully complete, entrainment in 20-day-old pups as compared with adult animals. The developmental interval when the photoperiod begins to entrain the core clock mechanism completely might thus occur around the time of weaning.

Reliability and methodological issues of power assessment during chest presses on unstable surface with different weights.

AIM: The study compares the reliability of peak power (Ppeak) and mean power in acceleration (Pmean acc) and entire concentric phase (Pmean total) of chest presses on the bench and unstable Swiss ball with different weights. METHODS: A group of 32 fit men performed over 2 testing sessions 3 trials of barbell chest presses on the bench and Swiss ball, without and with countermovement, with weights of 40, 60 and 80% 1RM. RESULTS: High values of correlation coefficients (above .80) and no significant differences between trials signify stability of measurement under both stable and unstable conditions. When chest presses were performed on the bench, ICC and SEM% values were in range .97 to .98 and 7.6 to 7.7%, respectively for Pmean total, .96 to .98 and 9.1 to 9.6%, respectively for Pmean acc, and .94 to .97 and 9.2 to 10.0%, respectively for Ppeak. Their values during chest presses on a Swiss ball ranged from .93 to .96 and 8.4 to 9.1%, respectively for Pmean total, from .87 to .90 and 11.7 to 12.2%, respectively for Pmean acc, and from .79 to .82 and 12.1 to 13.4%, respectively for Ppeak at weights of 40 and 60% 1RM, and from .70 to .76 and 17.6 to 19.8%, respectively at weight of 80% 1RM. CONCLUSION: Measurement of peak and mean power during unstable chest presses provides reliable data comparable to those obtained during bench presses under all conditions tested. However, peak values of power measured during unstable chest presses with weights ≥80% 1RM should be interpreted with caution.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

Protective effect of sulfoethylglucan against hexavalent chromium.

The effect of pretreatment with sulfoethylglucan (SEG) on the frequency of micronuclei and the liver alkaline phosphatase activity induced by potassium bichromate (Cr(VI)) in mice was evaluated. Simultaneous application of SEG and Cr(VI) decreased the frequency of micronuclei in bone marrow cells (P < 0.01) and the level of liver alkaline phosphatase activity in comparison to the Cr(VI) group. Pretreatment with SEG 24 h prior to the first Cr(VI) application resulted in a more pronounced decrease in the Cr(VI)-induced frequency of micronuclei. The mechanisms of the protective effects of sulfoethylglucan could be explained either by the formation of Cr ion complexes with sulfoethyl groups of glucan or by the scavenging ability of SEG to trap hydroxyl radicals.

Benzo[a]pyrene-enhanced mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

To study the suspected mechanism of the interaction between tobacco smoking and asbestos exposure in the modulation of cancer risk, the mutagenic potential of asbestos in combination with the tobacco smoke carcinogen benzo[a]pyrene (B[a]P) was examined in vivo in the rat lung. B[a]P was administered intratracheally in one set of experiments, or by two daily intraperitoneal injections in another set of experiments, to lambdalacI transgenic rats, together with 1, 2 or 4 x 2 mg amosite in one experiment. In the first experiment, the combined action of amosite and B[a]P caused a synergistic (superadditive) increase of mutation frequency in the lung, as compared to groups treated only with asbestos or B[a]P. In the second experiment, i.p. treatment with B[a]P did not significantly alter the mutation frequency induced by amosite, neither after 4 nor after 16 weeks of exposure. The B[a]P-DNA adduct levels were unaffected by amosite co-treatment in both experiments. We assume that the synergistic increase of mutation frequency after intratracheal treatment was due to the mitogenic activities of B[a]P and of amosite. In conclusion, our findings indicate that a weak and delayed mutagenic effect of amosite in rat lung observed in another study was strongly enhanced by the concomitant action of B[a]P. The striking enhancement effect of B[a]P may provide a basis for understanding the suspected synergism of smoking on asbestos carcinogenesis.

Mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic lambda-lacI (Big Blue) rats were intratracheally instilled with single doses of 1 or 2mg, or with four weekly doses of 2mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 x 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect.

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans.

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.

Effects of sodium diethyldithiocarbamate on type II pulmonary epithelial cells in vitro.

Dithiocarbamates (DDTC) are chemicals widely used in the form of pesticides, therapeutic and chelating agents, and scavengers. Since DDTC interfere with SH, Cu, and Zn enzymes due to chelating properties, it was of interest to clarify, in primary culture of type II alveolar pneumocytes, the effect of this compound upon enzymes of glutathione cycle, Cu, Zn-superoxide dismutase, and the membrane structure of cells. DDTC significantly inhibited the activity of superoxide dismutase and the activity of gamma-glutamyl transpeptidase, glutathione reductase, and alkaline phosphatase, whereas an increase in the activity of glutathione peroxidase was found. The membranes of pneumocytes type II were injured. Data show that DDTC adversely affected type II pneumocyte function and structure.

Diet containing fungal (1-->3)-beta-D-glucan derivative exhibits protective effects against DNA lesions induced in freshly isolated rat cells.

Dietary effect of water-soluble derivative - carboxymethyl chitin-glucan (CM-CG) on the level of DNA lesions induced by hydrogen peroxide (H2O2) was examined in ex vivo experiments. Lymphocytes, testicular cells, alveolar macrophages and epithelial II cells were isolated from Sprague Dawley rats fed a common or CM-CG enriched diet (200 mg/kg of body weight) during 21 days. Freshly isolated cells were in in vitro conditions exposed to H2O2 and the levels of DNA breaks were evaluated by single cell gel electrophoresis. A dose-dependent increase of DNA breaks was observed after treatment with hydrogen peroxide in all studied cell types. The levels of DNA breaks in cells isolated from CM-CG supplemented animals were lower compared to the levels of DNA breaks in cells isolated from animals fed a common diet. Intake of CM-CG enriched diet did not increase the level of DNA damage in different kinds of freshly isolated rat cells and equipped the cells with resistance to the treatment with hydrogen peroxide.

The chamber exposure of laboratory rats to metal oxides originating from metal producing industry.

Laboratory rats were exposed to the inhalation of dust from an agglomeration unit which is the greatest contributor to dust pollution in the vicinity of a mercury producing plant. The exposure lasted for 6 months (4 hours daily, 5 days per week), the concentration of aerosol in the chamber was 10 mg x m(-3). After finishing the exposure, the animals were examined and compared with the controls which were held under standard laboratory conditions. The number of alveolar macrophages was highly elevated (P< 0.001) in the exposed animals, Mg2+ ATPase activity in the heart muscle was decreased. The alanine aminotransferase activity in the serum was not changed, the aspartate aminotransferase was slightly enhanced. No differences in the frequency of abnormal sperm and in the frequency of polychromatic erythrocytes in bone marrow were detected.

Surface changes in type II pneumocytes isolated from rats during the cultivation.

Type II cells isolated from the rat lung were maintained in culture for 8 days. The activity of alkaline phosphatase and lectin binding properties were studied. The alkaline phosphatase activity and the number of lamellar bodies were continually decreasing during the studied time period. The profile of lectin binding (Maclura pomifera and Ricinus communis) did not change during the cultivation.

Seasonal molecular timekeeping within the rat circadian clock.

In temperate zones duration of daylight, i.e. photoperiod, changes with the seasons. The changing photoperiod affects animal as well as human physiology. All mammals exhibit circadian rhythms and a circadian clock controlling the rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN consists of two parts differing morphologically and functionally, namely of the ventrolateral (VL) and the dorsomedial (DM). Many aspects of SCN-driven rhythmicity are affected by the photoperiod. The aim of the present overview is to summarize data about the effect of the photoperiod on the molecular timekeeping mechanism in the rat SCN, especially the effect on core clock genes, clock-controlled genes and clock-related genes expression. The summarized data indicate that the photoperiod affects i) clock-driven rhythm in photoinduction of c-fos gene and its protein product within the VL SCN, ii) clock-driven spontaneous rhythms in clock-controlled, i.e. arginine-vasopressin, and in clock-related, i.e. c-fos, gene expression within the DM SCN, and iii) the core clockwork mechanism within the rat SCN. Hence, the whole central timekeeping mechanism within the rat circadian clock measures not only the daytime but also the time of the year, i.e. the actual season.

Ozone-induced micronuclei frequency in rat alveolar Type II cells.

The effect of ozone, a ubiquitous air pollutant, was tested on cultured pulmonary epithelial type II cells isolated from rats. After 40-hour culture, the cells were exposed for 6 h to 400 ppb of ozone or air. The number of micronucleated cells was counted after the exposure. In each group, 17000 cells were evaluated. The number of micronucleated cells was significantly increased in the ozone-exposed group (12.24 per 1000 cells) compared to the control group (5.00 per 1000 cells). The results showed the mutagenic effect of ozone exposure on alveolar type II cells, manifested in the increased frequency of their micronuclei.

Isolation and cultivation of lung epithelial type 2 cells. The effect of cadmium.

The effects of cadmium exposure on rat lung type 2 cells were evaluated by morphological and biochemical examinations. The results showed dose dependent reduction in the marker enzyme (alkaline phosphatase), changes in cell membranes and in the antioxidant status.

Effect of amikacin on cytotoxic activity and hydrophobicity of Acinetobacter baumannii.

Effect of amikacin at suprainhibitory (2 or 4 x MIC) or supra-subinhibitory concentrations (2 or 4 x MIC + 0.2 x MIC) on bacterial growth, cytotoxicity and cell surface hydrophobicity of three Acinetobacter baumannii strains was studied. Amikacin at suprainhibitory concentrations induced postantibiotic effects (PAEs; suppression of bacterial growth after short time exposure of bacteria to the antibiotic) against all A. baumannii strains. PAEs ranged from 1.2 to 2.9 h (2 x MIC) and 3.5 to 6.3 h (4 x MIC). Supra-subinhibitory concentrations of amikacin (2 x MIC + 0.2 x MIC) manifested a more significant delay of bacterial regrowth (PA SMEs) for two strains (6.6 or 7.5 h) in comparison with PAEs. One strain under these conditions as well as all strains treated with amikacin at 4 x MIC + 0.2 x MIC did not show any regrowth. Amikacin at all concentrations tested significantly reduced cytotoxic activity of A. baumannii evaluated on alveolar epithelial type II cells. Survival of type II cells after application of antibiotic-treated A. baumannii was in the range of 88 to 101% of the control cells. Cell surface hydrophobicity of amikacin-treated bacteria was practically unchanged varying between 94 and 100.9% as compared to the controls.

Stobadine pretreatment enhances glutathione peroxidase activity in the heart of irradiated mice.

The effect of pretreatment with stobadine (a novel drug with cardioprotective properties) on the activity of glutathione peroxidase was studied in the heart of mice after Co60 irradiation. Exposure to 6.5 Gy caused significant decrease in the activity of the enzyme (p < 0.01). Treatment with stobadine (70.07 mg/kg) 1 or 2 h before irradiation resulted in activity enhancement in comparison with the nonpretreated irradiated group (p < 0.01). We conclude that the radical scavenging mechanism may be involved in the protection exerted by stobadine. The results are in agreement with those obtained by the micronucleus test.

Selenium status, plasma zinc, copper, and magnesium in vegetarians.

Plasma zinc (Zn), copper (Cu), and magnesium (Mg) concentrations, copper/zinc ratio, and selenium (Se) status were studied in 44 vegetarians (22 males and 22 females) and their age- and sex-matched nonvegetarians in the Bratislava region (Slovakia). Vegetarians had statistically significant lower levels of plasma Zn and Cu than nonvegetarians, which may be the result of lower bioavailability of Zn and Cu from this type of diet. No differences in plasma Mg levels were found between vegetarians and nonvegetarians. Se status, as expressed by plasma and erythrocyte concentrations and plasma and erythrocyte glutathione peroxidase activities (GPx), was significantly lower in vegetarians when compared to nonvegetarians. In the series as a whole, there were significantly higher correlations between plasma and erythrocyte Se concentrations and between plasma and erythrocyte GPx activities. Significant positive correlations were also found between plasma Se concentrations and erythrocyte GPx activities, and between erythrocyte Se concentrations and erythrocyte GPx activities. A vegetarian diet does not provide a sufficient supply of essential antioxidant trace elements, like Zn, Cu, and especially Se. Se supplementation should be recommended to this risk group of the population.