Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.     

On the Interplay of Telomeres,Nevi and the Risk of Melanoma

The relationship between telomeres, nevi and melanoma is complex. Shorter telomeres have been found to be associated with many cancers and with number of nevi, a known risk factor for melanoma. However, shorter telomeres have also been found to decrease melanoma risk. We performed a systematic analysis of telomere-related genes and tagSNPs within these genes, in relation to the risk of melanoma, dysplastic nevi, and nevus count combining data from four studies conducted in Italy. In addition, we examined whether telomere length measured in peripheral blood leukocytes is related to the risk of melanoma, dysplastic nevi, number of nevi, or telomere-related SNPs. A total of 796 cases and 770 controls were genotyped for 517 SNPs in 39 telomere-related genes genotyped with a custom-made array. Replication of the top SNPs was conducted in two American populations consisting of 488 subjects from 53 melanoma-prone families and 1,086 cases and 1,024 controls from a case-control study. We estimated odds ratios for associations with SNPs and combined SNP P-values to compute gene region-specific, functional group-specific, and overall P-value using an adaptive rank-truncated product algorithm. In the Mediterranean population, we found suggestive evidence that RECQL4, a gene involved in genome stability, RTEL1, a gene regulating telomere elongation, and TERF2, a gene implicated in the protection of telomeres, were associated with melanoma, the presence of dysplastic nevi and number of nevi, respectively. However, these associations were not found in the American samples, suggesting variable melanoma susceptibility for these genes across populations or chance findings in our discovery sample. Larger studies across different populations are necessary to clarify these associations.     

OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA FROM LIVERS SHOWING CLOUDY SWELLING

Using succinate and α-ketoglutarate as substrates, oxidative phosphorylation has been measured in mitochondria isolated from livers showing cloudy swelling. This cellular change was obtained by injecting rats with S. typhi murium toxin and guinea pigs with diphtheria toxin. It has been found that phosphorylation associated with the oxidation of either of these substrates was partially inhibited in mitochondria from livers showing cloudy swelling, while the oxygen consumption was unchanged. Thus, the P:O ratios for both succinate and α-ketoglutarate were lower in mitochondria from treated animals than they were in normal mitochondria. Dephosphorylation of ATP was not significantly modified in mitochondria from livers showing cloudy swelling as compared with normal controls. No dephosphorylation of AMP and G-6-P was observed either in normal mitochondria or in mitochondria from treated animals.     

Multiple Sclerosis in the Mount Etna Region: Possible Role of Volcanogenic Trace Elements

Background

Trace elements have been hypothesised to be involved in the pathogenesis of Multiple Sclerosis and volcanic degassing is the major natural sources of trace elements. Both incidence of Multiple Sclerosis in Catania and volcanic activity of Mount Etna have been significantly increased during the last 30 years. Due to prevailing trade winds direction, volcanic gases from Etna summit craters are mostly blown towards the eastern and southern sectors of the volcano.

Objective

To evaluate the possible association between Multiple Sclerosis and exposure to volcanogenic trace elements.

Methods

We evaluated prevalence and incidence of Multiple Sclerosis in four communities (47,234 inhabitants) located in the eastern flank and in two communities (52,210 inhabitants) located in the western flank of Mount Etna, respectively the most and least exposed area to crater gas emissions.

Results

A higher prevalence was found in the population of the eastern flank compared to the population of the western one (137.6/100,000 versus 94.3/100,000; p-value 0.04). We found a borderline significantly higher incidence risk during the incidence study period (1980–2009) in the population of the eastern flank 4.6/100,000 (95% CI 3.1–5.9), compared with the western population 3.2/100,000 (95% CI 2.4–4.2) with a RR of 1.41 (95% CI 0.97–2.05; p-value 0.06). Incidence risks have increased over the time in both populations reaching a peak of 6.4/100,000 in the eastern flank and of 4.4/100.000 in the western flank during 2000–2009.

Conclusion

We found a higher prevalence and incidence of Multiple Sclerosis among populations living in the eastern flank of Mount Etna. According to our data a possible role of TE cannot be ruled out as possible co-factor in the MS pathogenesis. However larger epidemiological study are needed to confirm this hypothesis.     

Glutathione-mediated Antioxidative Mechanisms in Sunflower (<Emphasis Type="Italic">Helianthus Annuus</Emphasis> L.) Cells in Response to Cadmium Stress

Cadmium (Cd) homeostasis and detoxification in sunflower (Helianthus annuus L.) cells differing in Cd sensitivity/tolerance were studied by analyzing the glutathione-mediated antioxidant mechanism vis-à-vis phytochelatin biosynthesis in vitro. Calluses exposed to Cd-shock/-acclimatization (150μM) were assayed for oxidative stress, reduced glutathione (GSH), glutathione disulfide (GSSG), phytochelatins (PCs) and reactive oxygen species (ROS). Although Cd did not induce any oxidative stress in Cd-tolerant callus (TCd), it generated oxidative stress in Cd-shock callus (SCd) both in terms of lipid peroxidation and protein oxidation. GSH/GSSG ratio remained similar to control values in the cadmium-acclimatized calluses. However, after acute treatment, there was a decline in both GSH and GSSG levels in SCd with concomitant reduction in the GSH/GSSG ratio. Analysis of PCs was performed using HPLC and mass spectrometry methods. PC concentration in TCd were approximately twice those that in SCd, showing in both cases a 1:2:1 relative proportion for PC n = 2 (PC2): PC n = 3 (PC3): PC n = 4 (PC4). Calluses growing in the presence of Cd developed an increased resistance to paraquat oxidative stress generation. These results indicated that PCs synthesis was an important mechanism for Cd detoxification in sunflower calluses, but the capacity to grow in the presence of Cd is related to the tissues ability to maintain high intracellular levels of GSH.     

Cell envelope changes in Bifidobacterium animalis ssp. lactis as a response to bile

Bifidobacterium animalis ssp. lactis is a probiotic frequently used as adjunct culture in fermented dairy products. In order to ensure its proper function at the intestinal level, this bacterium has to be tolerant to physiological concentrations of bile. This study examined the influence of bile on the fatty acid composition and the membrane characteristics of B. animalis IPLA 4549 and its mutant with acquired resistance to bile, B. animalis 4549dOx. Bile adaptation triggers in B. animalis 4549dOx a decrease in membrane fluidity and in the protein : phospholipid ratio, as well as a shift in the fatty acid composition of the cell. Remarkably, the presence of bile in the growth medium induced similar changes in both B. animalis cells. Furthermore, transmission electron microscopy analysis showed that bile promotes a severe distortion of the cell surface. This study provides new insights of the action of bile on the cell envelope of bifidobacteria.