Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture

Bovine embryos cultured in serum-containing media abnormally accumulate lipids in the cytoplasm. This is well known to contribute to their higher susceptibility to cryopreservation and biopsied embryos are even further susceptible. We aimed to improve in vitro produced (IVP) embryos resistance to micromanipulation and cryopreservation by supplementing serum-containing media with trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA). The effect of t10, c12 CLA on lipid deposition and embryonic development was also tested. After in vitro maturation and fertilization (IVF day = D0), zygotes were cultured on granulosa cells + M199 + 10% serum + 100 μM GSH supplemented with 100 μM of t10, c12 CLA (CLA group, n = 1394) or without supplementation (control group, n = 1431). Samples of D7/D8 embryos were observed under Nomarsky microscopy for lipid droplets evaluation while others were biopsied and vitrified (group B-Control, n = 24; group B-CLA, n = 23). Non-biopsied embryos were also frozen (group NB-Control, n = 49; group NB-CLA, n = 45). Biopsied cells were used for embryo sex determination. Postwarming embryo survival and viability were determined at 0 and 24 h of culture, respectively. Supplementation of t10, c12 CLA did not influence cleavage, embryo sex ratio, D7/D8 embryo rate or morphological quality. CLA embryos had higher number of small lipid droplets (P ≤ 0.003) and a smaller (P < 0.001) fat embryo index being leaner (P = 0.008) than control embryos. Embryo postwarming survival was higher in B-CLA than in B-control group (95.0 ± 7.0% versus 62.5 ± 7.9%; P < 0.001). After 24 h of culture, the viability (expansion rate) of biopsied embryos and nonbiopsied embryos, cultured with t10, c12 CLA was higher than control embryos (B-CLA = 64.6 ± 4.4% and B-control = 27.5 ± 2.5%, P = 0.01; NB-CLA = 86.0 ± 3.5% and NB-Control = 68.6 ± 7.0%, P = 0.05). Results showed that supplying t10, c12 CLA to serum-containing media decreases embryo cytoplasmic lipid deposition during in vitro culture and significantly improves resistance of IVP embryos to micromanipulation and cryopreservation.     

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.     

No association between N7-methyldeoxyguanosine and 8-oxodeoxyguanosine levels in human lymphocyte DNA

To examine associations between two different classes of DNA damage that can occur through endogenous processes or exogenous exposures such as smoking, N7-methyldeoxyguanosine (N7-MedG) and 8-oxodeoxyguanosine (8-oxodG) levels were measured in lymphocyte DNA from 22 bronchoscopy patients. 8-OxodG and N7-MedG was detected in 100% and 91% of samples, respectively with 8-oxodG levels being approx 20 times higher (mean 8.39 ± 3.57 8-oxodG/10⁶dG versus 0.41 ± 0.33 N7-MedG/10⁶ dG) which provides an indication of the relative importance of the agents that induce oxidative DNA damage or alkylation damage. The sources of these genotoxic lesions remain to be established but N7-MedG and 8-oxodG levels were not correlated (r² < 0.01) suggesting that there is no association between alkylating agent and reactive oxygen species exposure, their metabolism and/or the DNA repair processes that can remove this DNA damage.     

Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods.The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10⁶ dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.     

Capsaicin-evoked bradycardia in anesthetized guinea pigs is mediated by endogenous tachykinins

The present study was done to characterize the effects of endogenous tachykinins on heart rate in urethane-anesthetized guinea pigs. Intravenous injection of capsaicin (32 nmol/kg) was used to evoke release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac sensory nerve fibers. Such injections caused a brief decrease in heart rate (− 37 ± 7 beats/min, n = 6) that was followed by a more prolonged increase (+ 44 ± 10 beats/min). Blood pressure was lowered by − 11 ± 2 mmHg. Bilateral vagotomy did not affect the chronotropic or depressor responses to capsaicin, but atropine (1 µmol/kg) nearly abolished the bradycardic response (− 8 ± 3 beats/min, n = 7). Combined blockade of NK₂ and NK₃ receptors, with SR48968 and SR14801 respectively, also caused a significant reduction of capsaicin-evoked bradycardia (− 14 ± 3 beats/min, n = 4) but did not affect bradycardia evoked by vagal nerve stimulation. Blockade of CGRP receptors eliminated capsaicin-evoked tachycardia and prolonged the capsaicin-evoked bradycardia. These findings suggest that capsaicin-evoked bradycardia in the anesthetized guinea pig is mediated by tachykinins that stimulate cardiac cholinergic neurons. This effect appears to be truncated by the positive chronotropic action of CGRP that is also released from cardiac afferents by capsaicin.     

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm², n=3) and that in the antrum (157±9 cells/cm², n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm², n=3), jejunum (2640±140 cells/cm², n=3), ileum (2070±42 cells/cm², n=3), proximal colon (2243±138 cells/cm², n=3) and distal colon (2633±376 cells/cm², n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.     

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.     

Body condition and protein supplementation positively affect periovulatory ovarian activity by non LH-mediated pathways in goats

Effects of rumen undegradable intake protein (UIP) supplementation on ovarian activity and serum insulin, GH, and LH were evaluated in goats having low or high body condition (BC). Goats with either low BC (n = 16, 28.7 ± 0.8 kg BW, BC = 2.1 ± 0.3) or high BC (n = 16, 38.4 ± 0.8 kg, BC = 3.2 ± 0.3) received, during 40-days, one of the two protein supplementation levels: without UIP or with UIP (120 g goat⁻¹ d⁻¹). Oestrus was synchronized with two i.m. doses of PGF₂, and jugular blood samples were collected from 36 to 42 h after the second prostaglandin injection at 15 min intervals. Serum concentrations of insulin, LH, and GH were measured The number of preovulatory follicles and the number of corpora lutea (CL) were evaluated by transrectal ultrasonography at 1 and 4 days after the second prostaglandin dose, respectively. Does with higher BC had more CL than those in the lower condition group (2.8 ± 0.2 versus 1.8 ± 0.2, P < 0.05). Similarly, goats receiving UIP supplementation had more follicles (2.6 ± 0.2 versus 1.9 ± 0.2, P < 0.05) and tended to have more CL (2.6 ± 0.2 versus 2.0 ± 0.2, P = 0.05) than does not receiving UIP. Neither BCS nor UIP supplementation affected serum GH or LH concentrations, pulsatility, or area under the curve. High BC does produced more insulin (1.92 ± 0.17 versus 0.81 ± 0.17 ng/mL, P < 0.01 ng/mL) than lower BC goats; the same for UIP-supplemented (1.69 ± 0.18 versus 1.04 ± 0.18, P < 0.05). Results suggest that the increased ovarian activity observed in both UIP-supplemented and higher BC goats was not the result of changes in LH or GH, suggesting effects at a local level, through changes in insulin in a non-GnRH-gonadotrophin dependent manner.     

Serum concentrations of Cu, Zn, Fe, Mo and Co in newborn lambs following systemic administration of Vitamin E and selenium to the pregnant ewes

This study was conducted to determine the effects of systemic administration of Vitamin E and selenium to pregnant ewes at the late stage of gestation on serum concentrations of Cu, Zn, Fe, Mo and Co in their offspring's. Pregnant Lori–Bakhtiari ewes (n = 14) were randomly assigned to receive Vitamin E and selenium (treatment group; n = 7) or distilled water (control group; n = 7), once 3 weeks and again 1 week before parturition. Blood samples were taken from jugular vein of lambs at parturition and once a week during the 4 week of age. Serum concentration of Fe, Cu, Zn, Mo and Co was determined by atomic absorption spectrophotometery.

At parturition, serum concentration of Cu, Zn, Mo and Co were identical in lambs of both groups, while the serum concentration of Fe (mean ± S.E.) was significantly higher in lambs of control (210.83 ± 9.05 μg/dl) than treatment group (140.71 ± 17.8 μg/dl).

From parturition to the forth weeks of age the serum concentrations of Fe and Cu were increased (P < 0.05) in lambs of treated group which was concomitant with a reduction in Zn concentration.

In conclusion, increase in serum concentrations of Cu and Fe during the first 4 weeks of age in lambs of ewes given vitamin E and selenium compound, could disturb the Zn:Cu and Zn:Fe ratios which in turn lead to zinc deficiency.     

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.     

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.     

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.     

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.     

The alternating current polarographic behavior and determination of lansoprazole and omeprazole in dosage forms and biological fluids

The alternating current (AC_t) polarographic behavior of lansoprazole (LNS) and omeprazole (OMP) was studied in Britton Robinson buffers (BRb) over the pH range 4.1–11.5. In BRb of pH 9.6 and 10.5, well-defined AC_t peaks were obtained for both LNS and OMP, respectively. The current–concentration plots were rectilinear over the ranges of 0.4–20 µg mL^− 1 and 0.2–10 µg mL^− 1 for LNS and OMP respectively. The minimum detection limits (S/N = 2) were 0.02 µg mL^− 1 (5.4 × 10^− 8 M) and 0.01 µg mL^− 1 (2.9 × 10^− 8 M) for LNS and OMP, respectively. The proposed method was successfully applied to the analysis of the two drugs in their commercial capsules. The average percent recoveries were favorably compared to those obtained by reference methods. Co-administered drugs such as naproxen and methotrexate did not interfere with the proposed method. The proposed method was further extended to the in-vitro determination of the lansoprazole in spiked plasma, the percentage recoveries was 98.47 ± 1.29 (n = 4). The pathway for the electrode reaction for both drugs involved reduction of the sulphonyl group into the corresponding thiol group at the Dropping Mercury Electrode. The advantages of the method were time saving and more sensitive than the other published voltammetric method. Yet The present study is the first report on the use of alterating current polarography (AC_t) in this respect.     

A thermodynamic and crystallographic study of complexes of the highly preorganized ligand 8-hydroxyquinoline-2-carboxylic acid

The metal ion complexing properties of the ligand HQC (8-hydroxyquinoline-2-carboxylic acid) are reported. The structures of [Zn(HQCH)₂] · 3H₂O (1) and [Cd(HQCH)₂] · 3H₂O (2) were determined (HQCH = HQC with phenol protonated). Both 1 and 2 are triclinic, space group , with Z = 2. For 1 a = 7.152(3), b = 9.227(4), c = 15.629(7) Å,  = 103.978(7)°, β = 94.896(7)°, γ = 108.033(8)°, R = 0.0499. For 2 a = 7.0897(5), b = 9.1674(7), c = 16.0672(11) Å,  = 105.0240(10)°, β = 93.9910(10)°, γ = 107.1270(10)°, R = 0.0330. In 1 the Zn has a distorted octahedral coordination geometry, with Zn–N of 2.00 and 2.15 Å, and Zn–O to the protonated phenolic oxygens of 2.431 and 2.220 Å. The structure of 2 is similar, with Cd–N bonds of 2.220 and 2.228 Å, with Cd–O bonds to the protonated phenolate oxygens of 2.334 and 2.463 Å. The structures of 1 and 2, and isomorphous Ni(II) and Co(II) HQC complexes reported in the literature, show very interesting short (<2.5 Å) O–O distances in H-bonds involving the protons on the coordinated phenolates and lattice water molecules. These are discussed in relation to the possible role of short low-energy H-bonds in alcohol dehydrogenase in mediating the transfer of the hydroxyl proton of the alcohol to an adjacent serine oxygen.

The formation constants for HQC are determined by UV–Visible spectroscopy at 25 °C in 0.1 M NaClO₄ with Mg(II), Ca(II), Sr(II), Ba(II), La(III), Gd(III), Zn(II), Cd(II), Ni(II), Cu(II), and Pb(II). These show greatest stabilization with metal ions with an ionic radius above 1.0 Å. This is as would be expected from the fact that HQC forms two five-membered chelate rings on complex-formation, which favors larger metal ions. The ligand design concept of using rigid aromatic backbones in ligands to achieve high levels of preorganization, and hence the high log K values (for a tridentate ligand) and strong metal ion selectivities observed for HQC, is discussed.     

Association between numbers of ovarian follicles in the first follicle wave and superovulatory response in ewes

The aim of this study was to examine the variability in the number of ovarian follicles in sheep and to determine if the average number of follicles per day influences the response to superovulation and resulting embryo quality. Ewes (n = 83) were synchronized and the number of follicles (≥2 mm diameter) in the ovaries were counted daily between Days 0 and 4 of the oestrous cycle using transrectal ultrasonography. Fourteen to 21 days later, 47 ewes were randomly chosen from the group and were treated with an intravaginal progestagen pessary for 12 days and superovulated with 1500 IU eCG administered as a single injection 10 days after sponge insertion. Ewes were mated and reproductive tracts were recovered after slaughter on Day 6 of pregnancy. The number of corpora lutea was counted, uterine horns were flushed and the morphology and developmental stage of the recovered oocytes/embryos was assessed. The mean daily number (±S.D.) (≥2 mm diameter) of follicles per ewe was 8.5 ± 2.8 (ranging between 3 and 16). After superovulation animals with few follicles (Low group: <8 follicles/day; n = 21) had fewer (P < 0.005) corpora lutea, total structures (unfertilized oocytes and embryos), good quality and total embryos compared to animals with many follicles (High group: ≥8 follicles/day; n = 23). No difference was found in the proportion of good quality embryos (relative to the total number; Low 0.68 ± 0.11 versus High 0.79 ± 0.08; P = 0.21) between the two groups, or the recovery rate, the number of unfertilized oocytes or the number of poor quality embryos per animal. We conclude that ewes with a higher number of follicles (≥8) during the first follicular wave had a better superovulatory response (in terms of corpora lutea and high quality embryos) 2–3 weeks later; however, there was no relationship between the number of follicles and the proportion of good quality embryos per animal.     

Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers

We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n = 20) originating from a common herd were assigned to three treatment groups using a 3 × 3 latin square design replicated in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received a commercially available organic mineral supplement (organic; n = 52); or (3) heifers received an all inorganic mineral supplement (inorganic; n = 55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.5) and control (1.6 ± 0.5) treated heifers than organic (0.4 ± 0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements.     

Reduction of 8-hydroxyguanine in human leukocyte DNA by physical exercise

We investigated the effect of physical exercise on the level of 8-hydroxyguanine (8-OH-Gua), a form of oxidative DNA damage, and its repair activity in human peripheral leukocytes. Whole blood samples were collected by venipuncture from 21 healthy male volunteers (10 trained athletes and 13 untrained men), aged 19-50 years, both before and after physical exercise. Trained athletes showed a lower level of 8-OH-Gua (2.4 ± 0.5/10⁶ Gua, p = 0.0032) before exercise when compared to that of untrained men (6.2 ± 3.5). The mean levels of 8-OH-Gua of untrained subjects decreased significantly (p = 0.0057) from 6.2 ± 3.5/10⁶ Gua (mean ± SD/10⁶ Gua) to 3.3 ± 1.4/10⁶ Gua after physical exercise. On the other hand, the mean levels of repair activity of untrained subjects significantly increased after exercise (p = 0.0093) from 0.037 ± 0.024 (mean DNA cleavage ratio ± SD) to 0.056 ± 0.036. In the trained athletes 8-OH-Gua level and its repair activity were not changed before and after the exercise. We also observed inter-individual differences in 8-OH-Gua levels and its repair activities. These results suggest that physical exercise causes both rapid and long-range reduction of oxidative DNA damage in human leukocytes, with individually different efficiencies.     

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.     

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.