Influence of PAHs in ambient air on chromosomal aberrations in exposed subjects: international study - EXPAH

The aim of this study was to determine the influence of carcinogenic polycyclic aromatic hydrocarbons (c–PAHs) in complex mixtures in ambient air on DNA damage (chromosomal aberrations) in occupationally exposed subjects measured as percent of aberrant cells (% AB.C.).

There were in total 203 exposed subjects and 150 respective controls in the whole project, allocated in three different European cities – Kosice (Slovakia), Prague (Czech Republic) and Sofia (Bulgaria). The studied population from Kosice (Slovakia) consisted of 106 subjects. From these 51 were exposed policemen and 55 were controls. The Czech population comprised 52 exposed policemen and 50 controls. In Bulgaria, there were two equally numerous exposed groups: 50 policemen and 50 professional bus drivers together with 45 controls. According to personal monitoring, policemen and bus drivers in the Bulgarian capital Sofia were exposed to the highest levels of c-PAHs amongst the exposed subject groups in the cities (45.3 ± 25.9 ng/m³ in policemen resp. 36.1 ± 31.6 ng/m³ in bus drivers in Sofia, 26.8 ± 39.8 ng/m³ for policemen in Kosice and 11.9 ± 11.2 ng/m³ for policemen in Prague), compared to the respective controls (24.9 ± 17.7 ng/m³ for controls in Sofia, 7.9 ± 3.8 ng/m³ for controls in Kosice and 6.2 ± 3.6 ng/m³ for controls in Prague).

We observed the following frequency of % AB.C. scored by conventional method: 2.60 ± 2.64 in exposed policemen and 2.14 ± 1.61 in controls in Kosice (p = n.s.); 2.33 ± 1.53 in exposed policemen and 1.94 ± 1.28 in controls in Prague (p = n.s.); 3.04 ± 1.64 in exposed policemen, respectively, 3.60 ± 1.63 in exposed bus drivers and 1.79 ± 0.77 in the control group in Sofia (p < 0.05, respectively, p < 0.05).

According to data from multiple regression analysis, and group comparison of smokers versus nonsmokers in Sofia also cigarette smoking (p = 0.055) and the age (p = 0.020) seem to play an important role within the aberrant cell formation in addition to the occupational c-PAHs exposure (p = 0.000). Smoking status was the modifying factor for % AB.C. in Kosice (p = 0.020) after multiple regression approach was employed.

In summary, we can say that subjects occupationally exposed to higher levels of c-PAHs in ambient air in Sofia are at greater genotoxic risk compared to those working indoors.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

Chromosomal aberrations by fluorescence in situ hybridization (FISH)--Biomarker of exposure to carcinogenic PAHs

The fluorescence in situ hybridization (FISH) technique with whole chromosome painting for chromosomes #1 and #4 was used to study the impact of air pollution containing higher concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in three European cities, Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). In each site were followed an exposed group, who were police officers or bus drivers who work usually through busy streets for at least 8 h, and a reference group, who spent more than 90% of their daily time indoors.

In Prague, a significant increase was observed in percentage of aberrant cells (% AB.C.) in the police officers compared to the reference group (0.33 ± 0.25 versus 0.24 ± 0.18, p < 0.05). In Kosice, the exposed group differed from reference in the endpoints F_G/100 1.52 ± 1.18 versus 1.12 ± 1.30, p < 0.05; % AB.C. 0.30 ± 0.19 versus 0.21 ± 0.20, p < 0.05; t/1000 3.91 ± 3.18 versus 2.84 ± 3.10, p < 0.05. In Sofia were followed two exposed groups: police officers and bus drivers. All FISH endpoints were significantly higher in police officers compared to reference group (F_G/100 1.60 ± 0.99 versus 0.82 ± 0.79, p < 0.01; % AB.C. 0.25 ± 0.14 versus 0.13 ± 0.13, p < 0.01; t/1000 4.19 ± 2.65 versus 2.13 ± 2.05, p < 0.05; rcp 1.46 ± 1.07 versus 0.70 ± 0.76, p < 0.05). In bus drivers compared to reference there was an increase in % AB.C. (0.25 ± 0.18 versus 0.13 ± 0.13, p < 0.05).

This is the first study when FISH method was used to analyze the impact of environmental air pollution. According to the original hypothesis it is expected that the most important group of chemicals responsible for the biological activity of air pollution represent c-PAHs.     

Repair competence assay in studies of the influence of environmental exposure to c-PAHs on individual susceptibility to induction of DNA damage

Previous results from studies performed in three European cities suggested a decrease in DNA repair efficiency observed in lymphocytes of subjects occupationally exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs). The aim of this study was to investigate whether a relationship between exposure to environmental c-PAHs and cellular vulnerability to the induction of DNA damage and its repair is confirmed in a pooled group of subjects from Prague, Košice and Sofia. The investigated pool consisted of 144 subjects occupationally exposed to environmental c-PAHs, who were municipal policemen or bus drivers. A control group of 115 matched individuals consisted of males unexposed at work to c-PAHs. The repair efficacy was evaluated by a comparison of the DNA damage detected by the single cell gel electrophoresis (SCGE) immediately after challenging the cells with X-ray irradiation, with residual damage (RD) being measured after an incubation period of 60 min. A stochastic concept for a mechanism of the interaction between DNA and various genotoxic exposures, was applied to analyze a relationship between exposure and biological effect in the studied sample. The outcome of the study confirms that the exposure to environmental c-PAHs or smoking cigarettes, significantly decreases DNA repair efficiency (repair efficiency in the pooled group of exposed individuals was 61.8 ± 11.8% versus 67.9 ± 9.9 in control, p < 0.001, and repair efficiency in group of smoking individuals was 63.0 ± 11.5% versus 65.9 ± 11.1 in nonsmokers, p < 0.005). The repair efficiency can be affected by a genetic polymorphism, such as subjects with a homozygous mutation in polymorphic CYP1A1_(Val/Val) enzyme, or slow NAT2 acetylators, who showed a considerably lower DNA repair efficiency (i.e. average repair efficiency in subgroups of fast acetylators was for the control subgroup 68.1% versus 66.5% in exposed subjects, while in the case of subgroups of slow acetylators, for the control group was 68.0% versus significantly less in the exposed subjects, 60.6%, p < 0.05). Smoking habits, or the diet's vitamin content, significantly affected the process. The results obtained confirm a potential value of the method as a biomarker of susceptibility in molecular epidemiology or preclinical studies, aimed at predicting susceptibility to various genotoxic exposures (environmental, occupational, therapeutic). To conclude, the research proved the influence of environmental c-PAHs, genotypes, and life styles on DNA damage and on its repair efficiency. Even low exposure to environmental c-PAHs altered DNA repair abilities of the subjects, which may result in an increased cancer risk. The findings confirm that c-PAHs should become pollutants that are subject to regulation.     

Air pollution by carcinogenic PAHs and plasma levels of p53 and p21(WAF1) proteins

We analyzed the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in ambient air on the plasma levels of p53 and p21^WAF1 proteins among city policemen, bus drivers and controls in three European cities: Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). p53 and p21^WAF1 proteins are key regulators of the cell cycle and are accepted as universal markers of genotoxic stress and DNA damage. In total 204 exposed subjects (100 smokers, 104 nonsmokers) and 152 controls (54 smokers, 98 nonsmokers) were analyzed. Personal exposure to c-PAHs was evaluated using personal samplers during the working shift. The levels of p53 and p21^WAF1 proteins were assessed by ELISA assay. There were no differences between the levels of either protein between exposed and controls, or smokers and nonsmokers, in any city. However, we observed significant differences in p53 plasma levels in all subjects regardless of the exposure status between the individual cities (median values: 5, 31, 234 pg/ml, p < 0.001, for Prague, Kosice and Sofia, respectively). The levels correspond to the differences in exposure levels to c-PAHs and benzo[a]pyrene (B[a]P) in the individual cities. A multiple linear regression analysis confirmed that c-PAHs exposure is a variable significantly affecting levels of both proteins in all locations. When all subjects were divided into the group exposed to below-median levels of c-PAHs and the group exposed to above-median levels of c-PAHs we found significantly higher p53, as well as p21^WAF1 levels in the above-median exposure group (p53, 167 pg/ml versus 25 pg/ml, p < 0.001; p21^WAF1, 2690 pg/ml versus 2600 pg/ml, p < 0.05). Among all subjects p53 plasma levels were positively correlated with p21^WAF1 levels, exposure to B[a]P, c-PAHs and levels of total DNA adducts; for p21^WAF1 levels we observed the positive correlation with cotinine, c-PAHs exposure, total and B[a]P-like DNA adduct levels. In conclusion our results suggest that p53 and p21^WAF1 proteins plasma levels may be useful biomarkers of c-PAHs environmental exposure.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part II: human cell lines

Principal aims of this study were at first, to find a relevant human derived cell line to investigate the genotoxic potential of PAH-containing complex mixtures and second, to use this cell system for the analysis of DNA adduct forming activity of organic compounds bound onto PM10 particles. Particles were collected by high volume air samplers during summer and winter periods in three European cities (Prague, Kosice, and Sofia), representing different levels of air pollution. The genotoxic potential of extractable organic matter (EOM) was compared with the genotoxic potential of individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as well as their artificial mixtures. Metabolically competent human hepatoma HepG2 cells, confluent cultures of human diploid lung fibroblasts (HEL), and the human monocytic leukemia cell line THP-1 were used as models. DNA adducts were analyzed by ³²P-postlabeling. The total DNA adduct levels induced in HepG2 cells after exposure to EOMs were higher than in HEL cells treated under the same conditions (15–190 versus 2–15 adducts/10⁸ nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5–3.7 adducts/10⁸ nucleotides). A direct correlation between total DNA adduct levels and c-PAH content in EOM was found for all EOMs in HepG2 cells incubated with 50 μg EOM/ml (R = 0.88; p = 0.0192). This correlation was even slightly stronger when B[a]P content in EOMs and B[a]P-like adduct spots were analyzed (R = 0.90; p = 0.016). As THP-1 cells possess a limited metabolic capacity for most c-PAHs to form DNA reactive intermediates and are also more susceptible to toxic effects of PAHs and various EOM components, this cell line seemed to be an inappropriate system for genotoxicity studies of PAH-containing complex mixtures. The seasonal variability of genotoxic potential of extracts was stronger than variability among the three localities studied. In HepG2 cells, the highest DNA adduct levels were induced by EOM collected in Prague in the winter period, followed by Sofia and Kosice. However, in the summer sampling period, the order was quite opposite: Kosice > Sofia > Prague. When the EOM content per m³ of air was taken into consideration in order to compare real exposures of humans to genotoxic compounds in all three localities, extracts from respirable dust particles collected in Sofia exhibited the highest genotoxicity regardless of the sampling period. The results indicate that most of DNA adducts detected in cells incubated with EOMs have their origin in low concentrations of c-PAHs representing 0.03–0.17% of EOM total mass. Finally, our results suggest that HepG2 cells have a metabolic capacity for PAHs similar to human hepatocytes and represent therefore the best in vitro model for investigating the genotoxic potential of complex mixtures containing PAHs among the three cell lines tested in this study.     

Chromosomal aberrations in tire plant workers and interaction with polymorphisms of biotransformation and DNA repair genes

We evaluated chromosomal aberrations in lymphocytes of 177 workers exposed to xenobiotics in a tire plant and in 172 controls, in relation to their genetic background. Nine polymorphisms in genes encoding biotransformation enzymes and nine polymorphisms in genes involved in main DNA repair pathways were investigated for possible modulation of chromosomal damage. Chromosomal aberration frequencies were the highest among exposed smokers and the lowest in non-smoking unexposed individuals (2.5 ± 1.8% vs. 1.7 ± 1.2%, respectively). The differences between groups (ANOVA) were borderline significant (F = 2.6, P = 0.055). Chromosomal aberrations were higher in subjects with GSTT1-null (2.4 ± 1.7%) than in those with GSTT1-plus genotype (1.8 ± 1.4%; F = 7.2, P = 0.008). Considering individual groups, this association was significant in smoking exposed workers (F = 4.4, P = 0.040). Individuals with low activity EPHX1 genotype exhibited significantly higher chromosomal aberrations (2.3 ± 1.6%) in comparison with those bearing medium (1.7 ± 1.2%) and high activity genotype (1.5 ± 1.2%; F = 4.7, P = 0.010). Both chromatid- and chromosome-type aberration frequencies were mainly affected by exposure and smoking status. Binary logistic regression analysis revealed that frequencies of chromatid-type aberrations were modulated by NBS1 Glu185Gln (OR 4.26, 95%CI 1.38–13.14, P = 0.012), and to a moderate extent, by XPD Lys751Gln (OR 0.16, 95%CI 0.02–1.25, P = 0.081) polymorphisms. Chromosome-type aberrations were lowest in individuals bearing the EPHX1 genotype conferring the high activity (OR 0.38, 95%CI 0.15–0.98, P = 0.045). Present results show that exposed individuals in the tire production, who smoke, exhibit higher chromosomal aberrations frequencies, and the extent of chromosomal damage may additionally be modified by relevant polymorphisms.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities—Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10⁸ nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7–757 adducts/10⁸ nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to −S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R = 0.83; p = 0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R = 0.94; p = 0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by ³²P-postlabelling.     

The morphology of the medial gastrocnemius in typically developing children and children with spastic hemiplegic cerebral palsy

We collected 3D ultrasound images of the medial gastrocnemius muscle belly (MG) in 16 children with spastic hemiplegic cerebral palsy (SHCP) (mean age: 7.8 years; range: 4–12) and 15 typically-developing (TD) children (mean age: 9.5 years; range: 4–13). All children with SHCP had limited passive dorsiflexion range on the affected side with the knee extended (mean ± 1SD: −9.3° ± 11.8). Scans were taken of both legs with the ankle joint at its resting angle (RA) and at maximum passive dorsiflexion (MD), with the knee extended. RA and MD were more plantar flexed (p < 0.05) in children with SHCP than in TD children.

We measured the volumes and lengths of the MG bellies. We also measured the length of muscle fascicles in the mid-portion of the muscle belly and the angle that the fascicles made with the deep aponeurosis of the muscle. Volumes were normalised to the subject’s body mass; muscle lengths and fascicle lengths were normalised to the length of the fibula.

Normalised MG belly lengths in the paretic limb were shorter than the non-paretic side at MD (p = 0.0001) and RA (p = 0.0236). Normalised muscle lengths of the paretic limb were shorter than those in TD children at both angles (p = 0.0004; p = 0.0003). However, normalised fascicle lengths in the non-paretic and paretic limbs were similar to those measured in TD children (p > 0.05). When compared to the non-paretic limb, muscle volume was reduced in the paretic limb (p < 0.0001), by an average of 28%, and normalised muscle volume in the paretic limb was smaller than in the TD group (p < 0.0001).

The MG is short and small in the paretic limb of children with SHCP. The altered morphology is not due to a decrease in fascicle length. We suggest that MG deformity in SHCP is caused by lack of cross-sectional growth.     

Ageing and dexamethasone associated sarcopenia: peculiarities of regeneration

The purpose of this study was to assess the development of ageing- and glucocorticoid-related sarcopenia on the level of myofibrillar apparatus, paying attention to the synthesis (SR) and degradation rate (DR) of contractile proteins, muscle strength, and daily motor activity. We also wanted to test the effect of ageing and dexamethasone (Dex) excess on the regeneration peculiarities of skeletal muscle autografts. Four and 30-month-old male rats of the Wistar strain were used. Ageing associated sarcopenia was calculated from gastrocnemius muscle relative mass decrease (from 5.6 ± 0.08 to 3.35 ± 0.04; p < 0.001). The SR of MyHC in old rats was 30% and actin 23% lower than in young rats. Dex treatment decreased SR of two main contractile proteins significantly in both age groups (p < 0.001) and increased DR during ageing from 2.11 ± 0.15 to 4.09 ± 0.29%/day (p < 0.001). Hindlimb grip strength in young rats was 5.90 ± 0.35 N/100 g bw and 2.64 ± 0.2 N/100 g bw (p < 0.001) in old rats.

Autografts of old rats have a higher content of adipose tissue 14.9 ± 1.1% in comparison with young rats 6.8 ± 0.51% (p < 0.001) and less muscle tissue 39.8 ± 2.6% and 48.3 ± 2.8%, respectively (p < 0.05).

Both, ageing and dex-caused sarcopenic muscles have diminished capacity for regeneration.     

Attenuated feeding responses to circadian and palatability cues in mice lacking neuropeptide Y

Neuropeptide Y (NPY) is a potent orexigenic peptide that is implicated in the feeding response to a variety of stimuli. The current studies employed mice lacking NPY (Npy−/−) and their wild-type (Npy+/+) littermates to investigate the role of this peptide in the feeding response to circadian and palatability cues. To investigate the response to a circadian stimulus, we assessed food intake during the 4-h period following dark onset, a time of day characterized by maximal rates of food consumption. Compared to Npy+/+ controls, intake of Npy−/− mice was reduced by 33% during this period (0.6 ± 0.1 g versus 0.9 ± 0.1 g; p ≤ 0.05). In contrast, intake did not differ between genotypes when measured over a 24-h period (3.7 ± 0.2 g versus 3.5 ± 0.3 g; p = ns). Furthermore, reduced dark cycle 4 h food intake in Npy−/− mice was not evident after a 24-h fast (1.4 ± 0.1 g for both genotypes; p = ns), despite a pronounced delay in the initiation of feeding (636 ± 133 s versus 162 ± 29 s; p ≤ 0.05). To investigate the role of NPY in the feeding response to palatability cues, mice were presented with a highly palatable diet (HP) for 1 h each day (in addition to having ad libitum access to chow) for 18 days. Npy+/+ mice rapidly increased daily HP intake such that by the end of the first week, they derived a substantial fraction of daily energy from this source (41 ± 3%). By comparison, HP intake was markedly reduced in Npy−/− mice during the first week (24 ± 7% of daily energy intake, p ≤ 0.05 versus Npy+/+), although it eventually increased (by Day 9) to values comparable to those of Npy+/+ controls. These experiments suggest that NPY contributes to the mechanism whereby food intake increases in response to circadian and palatability cues and that mechanisms driving food intake in response to these stimuli differ from those activated by energy restriction.     

Identification of UDP-glucuronosyltransferase 1A10 in non-malignant and malignant human breast tissues

UGT1A10 was recently identified as the major isoform that conjugates estrogens. In this study, real-time PCR revealed high levels of UGT1A10 and UGT2B7 mRNA in human breast tissues. The expression of UGT1A10 in breast was a novel finding. UGT1A10 and UGT2B7 mRNAs were differentially expressed among normal and malignant specimens. Their overall expression was significantly decreased in breast carcinomas as compared to normal breast specimens (UGT1A10: 68 ± 26 vs. 252 ± 86, respectively; p < 0.05) and (UGT2B7: 1.4 ± 0.7 vs. 12 ± 4, respectively; p < 0.05). Interestingly, in African American women, UGT1A10 expression was significantly decreased in breast carcinomas in comparison to normals (57 ± 35 vs. 397 ± 152, respectively; p < 0.05). Among Caucasian women, UGT2B7 was significantly decreased in breast carcinomas in comparison to normals (1.1 ± 0.5 vs. 13.5 ± 6, respectively; p < 0.05). Glucuronidation of 4-hydroxylated estrone (4-OHE₁) was significantly reduced in breast carcinomas compared to normals (30 ± 15 vs. 106 ± 31, respectively; p < 0.05). Differential down-regulation of UGT1A10 and UGT2B7 mRNAs, protein, and activity in breast carcinomas compared to the adjacent normal breast specimens from the same donor were also found. These data illustrate the novel finding of UGT1A10 in human breast and confirm the expression of UGT2B7. Significant individual variation and down-regulation of expression in breast carcinomas of both isoforms were also demonstrated. These findings provide evidence that decreased UGT expression and activity could result in the promotion of carcinogenesis.     

Fever and leukocytosis responses in goats to inhaled endotoxin are dose-dependent

Forty-five, weanling goats of mixed-sex, were randomly allotted to five treatment groups: no dust (n = 16), raw organic dust exposure for 15 min (n = 6), raw organic dust exposure for 1 h (n = 7), raw organic dust exposure for 3 h (n = 7), and raw organic dust exposure for 4 h (n = 9). Inhalation exposures were performed in a closed tent for the allotted times. The amount of endotoxin calculated to be in the dust was shown to be 26.9 μg/g. The amounts of dust introduced into the tent for each time period were 4.58 g/m³ of air for 15 min; 7.46 g/m³ of air for 1 h; 14.82 g/m³ of air for 3 h; and 40.60 g/m³ of air for 4 h. There was a significant increase in the white blood cell count in the animals dusted for 4 h, at 8 and 12 h following exposure. There was a significant decrease in the peripheral lymphocyte cell count following the 15 min exposure at 12 h post exposure, and there was a significant increase in the peripheral lymphocyte cell count following the 4 h exposure, 24 h after the exposure. There was a significant increase in the neutrophil cell count 8 and 12 h following the 4 h exposure, while there was a significant decrease in the neutrophil cell count 48 h following the 4 h exposure. There was a significant increase in the rectal temperatures of all goats receiving the 4 h dust exposure at all time periods (0 time, 4, 12, 24, and 48 h). There was a significant increase in the rectal temperatures of goats following the 1 h exposure, 8 h later. These results indicate that the larger the dose of inhaled endotoxin, the higher the resultant fever and leukocytosis.     

Glucocorticoids suppress vasopressin gene expression in human suprachiasmatic nucleus

Sleep impairment is one of the major side effects of glucocorticoid therapy. The mechanism responsible for this circadian disorder is unknown, but alterations in the suprachiasmatic nucleus (SCN), the biological clock of the human brain, are presumed to play a major role. In the present study, the amount of vasopressin mRNA (AVP mRNA) expression in the SCN was investigated in 10 glucocorticoid-exposed patients and 10 glucocorticoid free, age- and clock time of death-matched controls. The total amount of AVP mRNA, expressed as masked silver grains in the SCN, was two times lower in glucocorticoid-exposed patients (n = 10; 5115 ± 1314 μm²) than that in controls (n = 10; 11,021 ± 1408 μm²) (P = 0.006). There was also a 53% decrease in the total number of profiles in the SCN that expressed AVP mRNA in glucocorticoid-exposed patients (16,759 ± 3110) compared with those in controls (31,490 ± 3816) (P = 0.01). In conclusion, glucocorticoids have an inhibitory effect on AVP mRNA expression in the human SCN, which may be the biological basis of the circadian rhythm disturbances during glucocorticoid therapy.     

Clusterin in cerebrospinal fluid: analysis of carbohydrates and quantification of native and glycosylated forms

Clusterin is suggested to be involved in the pathogenesis of Alzheimer's disease. Clusterin expression is increased in brain tissue in affected regions of Alzheimer patients, and intense clusterin staining is found in both senile plaques and in neuronal and glia cells. In contrast, the cerebrospinal fluid level of clusterin in Alzheimer patients has, thus far, been found unchanged. Clusterin is a glycosylated protein, and an alteration of its glycosylation in Alzheimer's disease might influence accurate quantification in cerebrospinal fluid through interference of antibody binding to the protein. Using enzymatic deglycosylation of clusterin isolated from cerebrospinal fluid, we found that the carbohydrates attached to clusterin were of the N-linked type and sialic acids. Based on this finding, cerebrospinal fluid samples from Alzheimer patients (n = 99) and controls (n = 39) were analysed. The samples were treated with peptide: N-glycanase F, cleaving off N-linked carbohydrates, and clusterin was quantified before and after deglycosylation using a new sandwich enzyme-linked immunosorbent assay. Clusterin was significantly increased in Alzheimer patients, in both native (7.17 ± 2.43 AU versus 5.73 ± 2.09 AU; p = 0.002), and deglycosylated samples (12.19 ± 5.00 AU versus 9.68 ± 4.38 AU; p = 0.004). Deglycosylation led to increased measured levels of clusterin by 70% (p < 0.001) in Alzheimer patients and 67% (p < 0.001) in controls. These findings indicate that glycosylation of proteins may interfere with their quantification. The results show that clusterin is significantly increased in cerebrospinal fluid from Alzheimer patients as a group, supporting that clusterin might be involved in the pathogenesis of Alzheimer's disease. However, the individual clusterin levels overlap between the two groups, and thus cerebrospinal fluid clusterin measurement is not suitable as a biochemical marker in the diagnosis of Alzheimer's disease.     

Influence of short-term fasting during the luteal phase of the estrous cycle on ovarian follicular development during the ensuing proestrus of ewes

In order to study the effects of storage media and time of storage on the viability of unfertilized eggs of endangered Caspian brown trout (Salmo trutta caspius), the ova of this fish was stored in coelomic fluid and Cortland artificial media at 2–3 °C for 120 h. In this research, Cortland artificial medium was buffered with 20 mM of three different buffers: Hepes (C₈H₁₈N₂O₄S), Tris–HCl (C₄H₁₁NO₃–HCl) and sodium salt Hepes (C₈H₁₇N₂O₄SNa). The pH of these media were adjusted according to natural pH of coelomic fluid. The eggs that stored in these media fertilized at times 0 h (eggs fertilized prior to storage), 48, 72 and 120 h of post-stripping, using fresh and pooled sperm obtained from four to six males. According to the results of present study, time of storage showed a significant (p < 0.05) main effect on eyeing, hatching and eyed eggs mortality rates. Eyeing and hatching rates significantly (p < 0.05) decreased from 97.4 ± 2.1% and 95.1 ± 4.4% at time 0 (eggs fertilized prior to storage) to 77.9 ± 3% and 65.5 ± 5% after 120 h of storage. Within a similar period of time, eyed eggs mortality significantly (p < 0.05) increased from 2.4 ± 2.4% to 17.2 ± 3.9%. No significant (p > 0.05) main effect was found among media buffered with Tris–HCl (82.8 ± 3.2%, 73.4 ± 5.4%, 12.1 ± 4.5%), Hepes (88.2 ± 3.4%, 80.7 ± 5.5%, 9.3 ± 3.4%), sodium salt Hepes (77.8 ± 3.8%, 69.3 ± 5.7%, 12.2 ± 3.9%) and coelomic fluid (84.8 ± 3.8%, 77.7 ± 5.1%, 8.9 ± 2.7%) for eyeing, hatching and eyed eggs mortality rates. There was a negative correlation (r = −0.895, p < 0.001) between eyed eggs mortality and hatching rates. In conclusion, unfertilized eggs of endangered Caspian brown trout can be successfully stored for 48 h without significant loss of fertility. But, storage for 120 h results in the falling of hatching rate. In addition, no significant difference was found between viability rates of ova stored in coelomic fluid and artificial media, 120 h post-storage. It reveals that artificial media could be substituted for coelomic fluid as storage medium at least for 120 h in Caspian brown trout.     

Caprine luteinizing hormone isoforms during the follicular phase and anestrus

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p < 0.05) in A (12.0 ± 0.8%) as compared with F (5 ± 2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during A, and 3 ± 1% during F (p < 0.05). Predominant isoforms in A eluted in the pH range 9.99–9.0 (42 ± 3%) as compared to 7 ± 3% (p < 0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99–8.0, representing 55 ± 8%, while in A the proportion was 11 ± 2% (p < 0.01). Isoforms eluted at the pH range 7.9–7 represented a significantly greater proportion during A (5.0 ± 0.6%) as compared with F (3 ± 1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.     

Effects of early gentling and early environment on emotional development of puppies

In recent years much interest has been focused on early experiences and numerous studies have been carried out in order to understand their effects on the behaviour of adult animals. The aim of this preliminary study was to assess the effects of early gentling and early environment on the emotional stability of puppies. Forty-three dogs (16 females and 27 males) from seven litters were used. Four of these litters (in total 23 puppies) were raised in a professional breeding kennel, while the remaining litters lived in their owner's home, in a family atmosphere. Half of every litter was gently handled daily from the 3rd day postpartum until the 21st. In order to assess the puppies’ emotionality, an isolation test followed by an arena test were conducted on every puppy at the age of 8 weeks. Video recording of the tests allowed the measurement of each puppy's vocalization and exploratory activity. Data were analysed with the Newmann–Keuls’ test comparing four groups: non-handled puppies raised in family (NHF); handled puppies raised in family (HF); non-handled puppies raised in a professional breeding kennel (NHB); handled puppies raised in a professional breeding kennel (HB).

The results suggest that early environment strongly influences the emotional stability of puppies when put in isolation: latency to the first yelp was longer (p < 0.05) in the HB group (89.46 ± 66.42) compared to NHB (45.90 ± 52.76), NHF (13.10 ± 12.17) and HF (17.90 ± 14.32), and in the NHB compared to NHF and HF; duration of vocalizations was shorter (p < 0.05) in the HB (36.77 ± 54.16) and NHB group (72.80 ± 60.57) compared to NHF (149.78 ± 19.52) and HF (132.50 ± 45.24). Moreover, early gentling had a cumulative positive effect on the emotional development of puppies. For both environments, handled puppies were calmer. In fact, they showed longer latency to vocalize and handled puppies (HB = 119.00 ± 39.85; HF = 97.12 ± 33.56) spent significantly more time (seconds) in exploratory activity (p < 0.05) compared to the corresponding non-handled puppies (NHB = 64.90 ± 34.06; NHF = 57.00 ± 26.61).

Therefore, it is concluded that the deliberate inclusion of gentling during early puppyhood would be advantageous to the emotional development and welfare of the puppy, in particular for those at risk of limited or poor tactile stimulation in the early weeks.     

Biomarkers of air pollution exposure--a study of policemen in Prague

The effect of exposure to organic compounds adsorbed onto respirable air particles (<2.5microm) on DNA adducts in lymphocytes was studied in a group of non-smoking policemen (N=109, aged 35+/-0.9 years) working in the downtown area of Prague and spending >8h daily outdoors. Personal exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed on respirable particles was monitored in each subject for 48h before biological sampling. DNA adducts were analyzed by a (32)P-postlabelling assay, and total DNA adduct levels and B[a]P-like spots were determined. Further biomarkers included cotinine levels in urine to control for exposure to tobacco smoke, plasma levels of vitamins A, E and C and polymorphisms of metabolic genotypes (GSTM1, GSTP1, GSTT1, CYP 1A1-Msp I and Ile/Val, MTHFR, MS), DNA repair genotypes (XRCC1, hOGG1 and XPD exons 6 and 23) and the p53 gene (p53 Msp I and BstU I). All the biomarkers of exposure and effect were analyzed repeatedly during a period of one year at 2-3 month intervals (January, March, June, September 2004) to cover periods with high (winter) and low (summer) levels of air pollution. The highest personal exposure to c-PAHs was found in January (8.1+/-8.8ng/m(3)), while the other three sampling periods exhibited 3-4-fold lower c-PAH exposure. The total DNA adducts were only slightly elevated in January (2.08+/-1.60) compared to March (1.66+/-0.65), June (1.96+/-1.73) and September (1.77+/-1.77). B[a]P-like DNA adducts, however, were significantly higher in January than in the March and June sampling periods (0.26+/-0.14 vs. 0.19+/-0.12 and 0.22+/-0.13, respectively; p<0.0001 and p=0.017) indicating that c-PAH exposure probably plays a crucial role in DNA adduct formation in lymphocytes. No effect of individual metabololic or DNA repair genotypes on DNA adduct levels was observed. However, the combination of two genotypes encoding enzymes metabolizing c-PAHs - CYP 1A1 and GSTM1 - was associated with the levels of total and B[a]P-like DNA adducts under conditions of increased exposure to c-PAHs. Our study suggests that DNA adducts in the lymphocytes of subjects exposed to increased c-PAH levels are an appropriate biomarker of a biologically effective dose, directly indicating whether or not the extent of exposure to these compounds is related to an increased mutagenic and carcinogenic risk.     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.

Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.

The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.

Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.

Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).

These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.