Rumen microbial community and milk quality in Holstein lactating cows fed olive oil pomace as part in a sustainable feeding strategy.

The use of alternative feed ingredients from the Agro-industry could be an efficient tool to improve the sustainability of dairy cow production. Since the richness in polyphenols, olive oil pomace (OOP), produced during olive oil milling, seems a promising by-product to ameliorate milk's nutritional value. The aim of this study was to test the use of OOP produced by means of a new technology (biphasic with stone deprivation) in dairy cow feeding strategy to evaluate the effect on animal performances, rumen microbiota, biohydrogenation processes and milk quality by a multidisciplinary approach. Forty multiparous Italian-Friesian dairy cows, at middle lactation, were randomly allotted into two homogenous groups and fed respectively a commercial diet (CON) and the experimental diet (OOPD) obtained by adding OOP to CON as partial replacement of maize silage. The two diets were formulated to be isoproteic and isoenergetic. The same diets were tested also in an in vitro trial aimed to evaluate their rumen degradability (% DEG). The dietary supplementation with OOP did not affect DM intake, rumen % DEG and milk production. The milk's nutritional quality was improved by increasing several important functional fatty acids (FAs; i.e., linoleic acid, conjugated linoleic acid, oleic acid, vaccenic acid). This finding was related to a decrease in rumen liquor biohydrogenation rate of unsaturated FAs. The stochiometric relation between volatile FA production in the rumen and methanogenesis suggested that OOP lowers the methane potential production (CON = 0.050 mol/L vs OOPD = 0.024 mol/L, SEM = 0.005, P = 0.0011). Rumen microbiota and fungi community did not be strongly altered by OOP dietary inclusion because few bacteria were affected at the genus level only. Particularly, Acetobacter, Prevotellaceae_UCG-004, Prevotellaceae_UCG-001, Eubacterium coprostanoligenes, Lachnospira, Acetitomaulatum, Lachnospiraceae_NK3A20 group were more abundant with OOPD condition (P < 0.05). Data reported in this study confirm that the use of OOP in dairy cow feeding can be an interesting strategy to improve milk nutritional quality increasing functional FA content without compromising the rumen degradability of the diet or causing strong perturbation of rumen ecosystem and maintaining animal performances.

Hyperhomocysteinaemia and poor vitamin B status in chronic obstructive pulmonary disease.

BACKGROUND AND AIMS: Patients with chronic obstructive pulmonary disease (COPD) are at increased atherothrombotic risk. Preliminary findings have suggested that COPD patients may have increased plasma total homocysteine (tHcy), a cardiovascular risk factor often caused by a poor B vitamin status, but plasma levels of such vitamins were not measured. The aim of this study was to investigate hyperhomocysteinaemia in COPD and to determine whether it may be secondary to poor plasma concentrations of B vitamins. METHODS AND RESULTS: We performed a case-control, cross-sectional study of 42 patients with COPD and 29 control subjects. Folate, vitamin B12, vitamin B6, tHcy, renal function, C-reactive protein, blood gases and lipids were measured in patients and controls. COPD patients had higher plasma tHcy (median: 13.9mumol/l, interquantile range [IQR]: 12.1-18.5 versus 11.5, IQR: 10.1-14, p=0.002) and lower circulating folate (median: 2.5ng/ml, IQR: 1.2-3.3 versus 2.8, IQR: 2.1-4 of controls, p=0.03) than controls had. Compared to the control group, COPD was associated with higher tHcy concentrations also after adjusting for smoking, heart failure, renal function and C-reactive protein with logistic regression analysis (OR 1.36, 95% CI 1.06-1.72, p=0.01). In the COPD group, low levels of folate (beta=-0.27, p=0.02) and vitamin B12 (beta=-0.24, p=0.04), and hypertriglyceridaemia (beta=0.580, p<0.0001) were independent predictors of the presence of high tHcy concentrations in a multiple linear regression model (adjusted R(2)=0.522). CONCLUSION: COPD patients have a poor B vitamin status and, as a consequence, increased tHcy. These abnormalities may contribute to the COPD-related atherothrombotic risk.

Impact of inherited polymorphisms in glutathione S-transferase M1, microsomal epoxide hydrolase, cytochrome P450 enzymes on DNA, and blood protein adducts of benzo(a)pyrene-diolepoxide.

The benzo(a)pyrene (BaP) metabolite benzo(a)pyrenediolepoxide (BPDE) is strongly implicated as a causative agent of lung cancer. To assess the risk of exposure to BaP, we made a combined analysis of levels of BPDE adducts to hemoglobin (Hb), serum albumin (SA), and lymphocyte DNA in 44 patients with incident lung cancer, as a prototype of a population mainly exposed to tobacco-derived BaP. We also investigated whether genetic polymorphisms of cytochrome P450IA1 (CYPIA1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase M1 (GSTM1), which are involved in BaP metabolism, can be determinants of adduct formation. BPDE-Hb, BPDE-SA, and BPDE-DNA adducts were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry to achieve high specificity and sensitivity. Individuals with detectable Hb adducts were positive for SA adducts but not vice versa, suggesting that BPDE-Hb adducts are less informative indicators of BaP exposure. Using PCR methods on DNA, we characterized GSTM1 deletion, CYPIA1 MspI and exon 7 valine variants, and mEH polymorphisms at amino acid positions 113 (EH3) and 139 (EH4). Levels of BPDE adducts were no different among CYPIA1, mEH, and GSTM1 genotypes. However, individuals with measurable BPDE-SA adducts were CYPIA1 variant carriers more frequently (P = 0.03). There was a slightly higher percentage of DNA detectable adducts in subjects with CYPIA1 exon 7 valine polymorphism. When subjects were classified by both polymorphisms on the mEH gene, those with two slow alleles (EH3 homozygous mutated) and no fast alleles (EH4 homozygous wild type) had a lower frequency of BPDE-SA adducts and no DNA adducts (P = 0.06). These results are based on a small number of observations thus far, but this exploratory study suggests that CYPIA1 and mEH variants might have an impact on BPDE exposure markers such as BPDE-SA adducts. Chemical specificity in adduct measurements is important to identify the biomarkers that reflect BaP exposure more accurately.

Seasonal effect on airborne pyrene, urinary 1-hydroxypyrene, and benzo(a)pyrene diol epoxide-hemoglobin adducts in the general population.

Exposure to airborne polycyclic aromatic hydrocarbons (PAHs) in 65 employees (40 sampled both in summer and winter, 15 sampled in summer only, and 10 sampled in winter only) with no occupational exposure to PAHs was assessed by measuring: personal exposure to pyrene, urinary excretion of 1-hydroxypyrene (1-OHP), and benzo(a)pyrene diol epoxide adducts to hemoglobin (BPDE-Hb). Overall, office employees were exposed to significantly higher levels of pyrene in winter (4.54 +/- 2.35 ng/m3, mean +/- SD) than in summer (1.67 +/- 1.92 ng/m3, mean +/- SD; P < 0.001), but no such seasonal variability was observed in 1-OHP excretion. Tobacco smoking was the major determinant of 1-OHP excretion. BPDE-Hb adducts were measured by gas chromatography-mass spectrometry as benzo(a)pyrene tetrols (BPT) released from adducted hemoglobin. In the 65 employees analyzed, mean BPT levels +/- SD were higher in winter (0.14 +/- 0.38 fmol/mg Hb) than summer (0.031 +/- 0.022 fmol/mg Hb). This difference was not statistically significant, probably because of the small proportion of subjects with detectable adducts (11% in summer and 16% in winter). BPDE-Hb adducts were not significantly associated with sex, age, diet, smoking habits, or with pyrene levels and 1-OHP excretion. This is the first report providing reference BPDE-Hb adduct values for the general population not occupationally exposed to environmental PAHs and shows a tendency to seasonal variability, with higher BPT levels in winter when environmental PAHs are also high.

Metabolic gene polymorphism frequencies in control populations.

Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.

Control of nifH transcription in Azospirillum brasilense: involvement of NifA and of cis-acting sequences.

The regulatory sequences of Azospirillum brasilense Sp7 nifH gene were fused with the cam reporter gene and used for studying the factors controlling nifH transcription. A DNA sequence, downstream the ATG codon of nifH, that could be involved in the negative regulation of nifH transcription, was identified. The effect of 1 and 2 mM of ammonium on the transcription of the A. brasilense nifH gene and on the nitrogenase activity, in the presence of the Klebsiella pneumoniae NifA protein, was examined.

Effect of butylated hydroxyanisole added in vitro or administered to rats on N,N-dibutylnitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine metabolism by post-mitochondrial supernatant of liver homogenates.

The effect of butylated hydroxyanisole (BHA) on P-450-dependent omega-hydroxylation of N,N-dibutylnitrosamine (NDBA) to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), and the further oxidation of BBN to N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) by the alcohol/aldehyde dehydrogenase system was investigated using the post-mitochondrial supernatant of liver homogenates (S9) from acutely and chronically BHA pretreated animals or S9 fractions from untreated rats with BHA added. Acute oral BHA (50 and 250 mg.kg-1) did not change NDBA omega-oxidation, which was reduced by 35% only when the compound was administered 0.5% in the diet for 3 weeks. BCPN formation from BBN was unaffected by acute and chronic BHA pretreatment. In order to verify whether BHA or its metabolite(s) had a direct effect on NDBA and BBN oxidation, the compound was added to S9 fractions from untreated rats at various concentrations. Only when BHA concentrations were equimolar or in a 10-fold molar excess to the substrate concentration, we observed 30-50% inhibition of BBN formation and a reduced BCPN formation (60-80% of control values), from BBN. Thus, only at very high BHA concentrations could we confirm the inhibition of P-450-dependent mixed function oxidase and alcohol dehydrogenase activities involved in the metabolism of NDBA and BBN.

Effect of butylated hydroxyanisole on in vitro and in vivo nitrosation of dibutylamine.

The effect of the food additive butylated hydroxyanisole on the nitrosation of dibutylamine was studied in vitro and in vivo, in rats. At the highest concentration tested, butylated hydroxyanisole significantly inhibited the in vitro formation of N-nitrosodi-n-butylamine from dibutylamine and nitrite by 35%. This effect was not observed in animals given dibutylamine, NaNO2 and butylated hydroxyanisole by gavage in the same ratio that produced an effect in vitro. The oral administration of butylated hydroxyanisole to rats given 0.5% nitrate in the drinking water and dibutylamine (0.38 mmol/kg 3 times/day p.o.) was also with no effect, although the experimental model used proved suitable for studying the modulating effect on endogenous nitrosation of different chemicals such as ascorbic acid or potassium thiocyanate. The discrepancy between in vitro and in vivo results is discussed.

Aromatic DNA adducts in lymphocytes of humans working at high and low traffic density areas.

Aromatic DNA adduct levels were determined by the 32P-postlabelling assay in lymphocytes isolated from newspaper vendors working at urban high traffic areas (n = 31) and suburban low traffic areas (n = 22) in Milan, Italy. The DNA adduct levels ranged from 0.7 to 6.7/10(8) nucleotides, while most of them were between 1.0 and 3.0/10(8) nucleotides. No difference was found between the DNA adduct levels of the high-exposed group (2.2/10(8) and the low-exposed group (2.2/10(8). The heavy smokers (n = 8) had 23% higher DNA adduct level (2.7/10(8)) than the non-smokers (n = 37, 2.2/10(8) (P = 0.27), but no correlation was found between the adduct level and the number of cigarettes/day. Analysis of variance of the DNA adduct levels among the 14 pairs of individuals working at the same news-stands revealed little effect of the environmental air exposure on the DNA adduct level.

Effect of acute and chronic butylated hydroxyanisole administration on in vivo glucuronidation of N-nitrosobutyl(4-hydroxybutyl)amine in rats.

The urinary metabolic pattern of N-nitrosobutyl(4-hydroxybutyl)amine (NB4HBA) administered ip at a dose of 5 mg/kg body weight was studied in animals either pretreated with butylated hydroxyanisole (BHA) as a single oral dose of 50 or 250 mg/kg, or fed a diet containing 0.1 or 0.5% BHA. The 24-hr urinary excretion of NB4HBA, its glucuronic acid-conjugate (NB4HBA-G) and N-nitrosobutyl(3-carboxypropyl)amine (NB3CPA) in control rats were 0.12, 0.75 and 30% of the administered dose, respectively, and were not changed after a single oral dose of 50 mg BHA/kg. NB4HBA-G was significantly reduced in the urine of rats given 250 mg BHA/kg. In vitro assays carried out using rat-hepatic microsomal preparations as the source of the enzyme UDP-glucuronyl transferases (GT) and NB4HBA as the substrate, suggest that a competition between NB4HBA and BHA for the same enzyme may be the cause of the decreased NB4HBA-G excretion observed in vivo. A fourfold increase in NB4HBA-G urinary excretion was observed after chronic 0.5% BHA feeding; moreover, the glucuronic acid-conjugate of NB3CPA (NB3CPA-G), which was not detected in the controls or after acute BHA treatment, appeared in the urine of rats given dietary BHA for 3 wk, accounting for about 10% of the administered NB4HBA. In vitro experiments indicate that the increased glucuronides excretion may be the result of an elevated hepatic GT activity.

Kinetics of 3-tert-butyl-4-hydroxyanisole (BHA) in man.

High-resolution capillary gas chromatography-mass spectrometry with selective ion monitoring and using deuterated 3-tert-butyl-4-hydroxyanisole (BHA) as an internal standard was used to measure BHA in the plasma and urine of human volunteers after oral administration of 30 or 5 mg of the compound in olive oil. Pharmacokinetic studies showed similar plasma-concentration profiles in subjects treated with either level of BHA. About 20% of the administered dose was excreted as BHA glucuronide in the urine within the first 24 hr.

Carcinogen-DNA adducts as tools in risk assessment.

Genotoxic chemicals are known to react with DNA either directly or after metabolic activation to form adducts, a step thought to be relevant with respect to chemical carcinogenesis. Evaluation of cancer risk due to exposure to chemicals requires information about the internal dose which depends on individual variation in rates of metabolic activation and detoxification. The presence and the amount of specific DNA adducts provide a good indication of chemical exposure and genetic damage resulting from exposure to carcinogens and account for some of the factors affecting individual susceptibility to cancer. Analysis of DNA adducts requires that the sensitivity of the methods be sufficiently high to allow the detection of about 1 adduct/109 normal nucleotides. Most suitable methods are based on 32P-postlabelling, immunoassays or physico-chemical techniques such as HPLC coupled to synchronous fluorescence spectroscopy or gas chromatography-mass spectrometry. These methods have been used to assess human exposure to a variety of chemical carcinogens including polycyclic aromatic hydrocarbons, aromatic amines, heterocyclic aromatic amines or aflatoxins. In some instances, the use of DNA-adducts has given accurate estimates of risk.

Orally dissolved ketanserin in acute treatment of hypertensive patients: a controlled study.

The effectiveness of 40 mg ketanserin dissolved in the mouth in reducing blood pressure was studied in 17 hypertensive patients. Comparison with a placebo group has shown that in the first 10 min the blood pressure trend was similar for both groups; a significant fall was recorded 20 and 60 min after administration for systolic blood pressure and after 60 min for diastolic blood pressure. Therefore, orally dissolved ketanserin decreases blood pressure more rapidly than with standard oral consumption, and may be used when a rapid progressive decrease in blood pressure is required. This antihypertensive effect might be more rapid by administering more soluble tablets or ketanserin in solution.

Immunomodulatory effects of occupational exposure to mancozeb.

The effects of occupational exposure to ethylene-bis-dithiocarbamate of manganese and zinc on the immune system were evaluated in a group of mancozeb-exposed manufacturers and controls. The immune system tests revealed the following: (a) lymphocyte proliferative responses triggered by different activators and mitogen-induced IL-2 production were higher in exposed subjects than in controls; (b) production of monocyte/macrophage-derived IL-1 and polyclonal IgG and IgM, by beta-lymphocytes, did not differ between exposed subjects and controls; (c) percentages and absolute numbers of total T-cells, T-helper cells, T-suppressor/cytotoxic cells, activated T-cells, total beta-cells, and natural killer cells were similar in exposed subjects and controls; (d) serum immunoglobulin classes and complement fractions were within the range of normality; and (e) rheumatoid factor and non-organ-specific serum autoantibodies were absent in exposed and control subjects. An increase in T-cell functional response was found in the exposed group, suggesting a slight immunomodulator effect of mancozeb in conditions of low-level, prolonged occupational exposure.

Acute captopril treatment in elderly hypertensive patients: a controlled study.

We studied the effectiveness of 50 mg captopril in reducing blood pressure in 27 elderly hypertensive patients. Comparison with a placebo group has shown a significant difference 20 and 60 min after administration (dissolved in the mouth). A significant decrease in blood pressure was found at each observation time (10, 20 and 60 min) in the captopril-treated group, whereas in the placebo group there was no significant fall after the first 10 min. Therefore, captopril dissolved in the mouth decreases blood pressure more rapidly than with standard oral consumption, and can be considered a first-choice drug when a rapid decrease in blood pressure is desired.

The genomic and proteomic blueprint of mouse megakaryocytes derived from embryonic stem cells.

BACKGROUND: Platelets are specialized cells, produced by megakaryocytes (MKs) in the bone marrow, which represent the first defense against hemorrhage. There are many diseases where platelet production or function is impaired, with severe consequences for patients. Therefore, new insights into the process of MK differentiation and platelet formation would have a major impact on both basic and clinical research. OBJECTIVES: Embryonic stem (ES) cells represent a good in vitro model to study the differentiation of MKs, with the possibility of being genetically engineered and constituting an unlimited source of MKs. However, lack of knowledge about the molecular identity of ES-derived MKs (ES-MKs) may prevent any further development and application of this model. METHODS: This paper presents the first comprehensive transcriptional and proteome profile analyses of mouse ES-MKs in comparison with MKs derived from mouse fetal liver progenitors (FL-MKs). RESULTS: In ES-MKs we found a down-regulation of cytoskeleton proteins, specific transcription factors and membrane receptors at both transcriptional and protein levels. At the phenotypic level, this molecular blueprint was displayed by ES-MKs' lower polyploidy, lower nuclear/cytoplasm ratio and reduced capacity to form proplatelets and releasing platelets. CONCLUSIONS: Overall our data demonstrate that ES-MKs represent a useful model to clarify many aspects of both MK physiology and pathological conditions where impaired MK functions are related to defective MK development, as in inherited thrombocytopenias and primary myelofibrosis.

Environmental tobacco smoke and risk of respiratory cancer and chronic obstructive pulmonary disease in former smokers and never smokers in the EPIC prospective study.

OBJECTIVES: To investigate the association between environmental tobacco smoke, plasma cotinine concentration, and respiratory cancer or death. DESIGN: Nested case-control study within the European prospective investigation into cancer and nutrition (EPIC). PARTICIPANTS: 303,020 people from the EPIC cohort (total 500,000) who had never smoked or who had stopped smoking for at least 10 years, 123,479 of whom provided information on exposure to environmental tobacco smoke. Cases were people who developed respiratory cancers or died from respiratory conditions. Controls were matched for sex, age (plus or minus 5 years), smoking status, country of recruitment, and time elapsed since recruitment. MAIN OUTCOME MEASURES: Newly diagnosed cancer of lung, pharynx, and larynx; deaths from chronic obstructive pulmonary disease or emphysema. Plasma cotinine concentration was measured in 1574 people. RESULTS: Over seven years of follow up, 97 people had newly diagnosed lung cancer, 20 had upper respiratory cancers (pharynx, larynx), and 14 died from chronic obstructive pulmonary disease or emphysema. In the whole cohort exposure to environmental tobacco smoke was associated with increased risks (hazard ratio 1.30, 95% confidence interval 0.87 to 1.95, for all respiratory diseases; 1.34, 0.85 to 2.13, for lung cancer alone). Higher results were found in the nested case-control study (odds ratio 1.70, 1.02 to 2.82, for respiratory diseases; 1.76, 0.96 to 3.23, for lung cancer alone). Odds ratios were consistently higher in former smokers than in those who had never smoked; the association was limited to exposure related to work. Cotinine concentration was clearly associated with self reported exposure (3.30, 2.07 to 5.23, for detectable/non-detectable cotinine), but it was not associated with the risk of respiratory diseases or lung cancer. Frequent exposure to environmental tobacco smoke during childhood was associated with lung cancer in adulthood (hazard ratio 3.63, 1.19 to 11.11, for daily exposure for many hours). CONCLUSIONS: This large prospective study, in which the smoking status was supported by cotinine measurements, confirms that environmental tobacco smoke is a risk factor for lung cancer and other respiratory diseases, particularly in ex-smokers.

Revision of species delineation in the genus Ectothiorhodospira.

When the type strains and other strains of the six currently defined species of the genus Ectothiorhodospira were examined by DNA-DNA reassociation and RFLP of 16S/23S rDNA (ribotype), only four genospecies could be found. The possibility of defining taxonomically meaningful species corresponding to these four genospecies was investigated by combining DNA relatedness and ribotype data with other genotypic and phenotypic characters already described in the literature, an approach known as polyphasic taxonomy. Following this comparison, the type strain and another strain of Ectothiorhodospira vacuolata were found to be very similar to the type strain of Ectothiorhodospira shaposhnikovii and have been transferred to this latter species. Also, the type strain of Ectothiorhodospira marismortui and another previously unidentified strain were found to be very similar to the type strain of Ectothiorhodospira mobilis and have been transferred to this latter species. Due to the limited degree of reciprocal DNA relatedness, strains belonging either to Ectothiorhodospira marina or to Ectothiorhodospira haloalkaliphila are still considered as belonging to separate species, even though they show a remarkable phenotypic similarity. This revision has led to the delineation of only four species in the genus Ectothiorhodospira, namely E. mobilis, E. shaposhnikovii, E. marina and E. haloalkaliphila. E. vacuolata is recognized as a junior synonym of E. shaposhnikovii and E. marismortui as a junior synonym of E. mobilis.

Effect of butylated hydroxyanisole on the metabolism of N-nitrosodi-n-butylamine and N-nitrosobutyl(4-hydroxybutyl)amine by rat hepatic S9 preparations in vitro.

N-Nitrosodi-n-butylamine (NDBA), the NADPH generating system and various concentrations of butylated hydroxyanisole (BHA) added to rat hepatic S9 fractions resulted in a significant drop (30-50%) in N-nitrosobutyl(4-hydroxybutyl)amine (NBHBA) formation and a consequent rise in the amount of substrate recovered unchanged. When NBHBA and NAD+ were incubated with BHA and S9 fractions, the amount of N-nitrosobutyl(3-carboxypropyl)amine (NBCPA) was decreased by 20-40%, and the amount of unmetabolized NBHBA increased.