P. Ormas

A clonal cell line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1.

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1--1.0-8.0 microM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2-4 microM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC50 at 48 h = 12.00 +/- 2.66 microM; MTT: CC50 at 72 h = 20.42 +/- 7.30 microM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.

Identification of Functional α-Adrenoceptor Subtypes in the Bovine Female Genital Tract During Different Phases of the Oestrous Cycle

The concentration and functionality of the α-adrenoceptor (α-AR) subtypes in the genital tract of cyclic heifers were investigated. In each tissue sample, a single class of α1-ARs was observed, whereas two distinct classes of α2-ARs were discriminated: low-affinity (LA) and high-affinity (HA) α2-ARs. Statistical analysis showed the presence of significantly (p < 0.05) higher concentrations of all α-AR subtypes in the follicle than in the corpus luteum. No significant differences were found in the ovary or myometrium between the luteal and follicular phases. In the ovary, the density of α1-ARs was significantly (p < 0.05) higher than that of α2-ARs. By contrast, there were significantly (p < 0.05) more α2-ARs than α1-ARs in the myometrium. As far as α2-ARs are concerned, LA α2-ARs were significantly (p < 0.05) higher than HA α2-ARs in all tested tissues. Competition studies suggested that the rank order of potency of antagonists for α1-ARs was prazosin > phentolamine > yohimbine, whereas for α2-ARs the order of potency was yohimbine ≥ phentolamine > prazosin. Functional assays performed on myometrium showed that noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions only in dioestrus and pro-oestrus preparations and that clonidine was more effective than phenylephrine as a contractile agent. It appeared that there were no significant modifications in α-AR affinity or concentration during the different stages of bovine oestrous cycle, whereas the uterine spontaneous activity and the responsiveness to α-adrenergic stimulation was strongly influenced by hormonal levels. The modifications of uterine contractility observed during the oestrous cycle may be related to modifications induced in the transductional mechanisms of α-ARs.

A rapid and simple method for the separation of pure lymphocytes from horse blood.

A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further centrifugation at 250 xg for 3 min (r.t.) of heparinized lymphocyte preparations removed an average of 87.39% (n = 15) contaminating platelets. A suspension of 85.96 +/- 2.20% pure granulocytes (93.23 +/- 1.74% neutrophils; n = 14) with minimal contamination by erythrocytes and high viability (93.11 +/- 1.26%) was obtained by performing a flash red blood cell lysis on the white-greyish layer resulting from the centrifugation of the heparinized blood samples. Among the several methods available, the procedure described herein is easy, rapid, cheap and reproducible.

Effects of endotoxin and influence of cyclooxygenase-2 on β-adrenergic mediated relaxation in isolated equine digital artery.

The effects of endotoxin on β-adrenergic-mediated relaxation were investigated in the equine digital artery (EDA). Possible involvement of cyclooxygenase-2 (COX-2) in endotoxin-induced effects and basal EDA β-adrenoceptor functionality was also evaluated. Endothelium-intact (e(+)) and/or -denuded (e(-)) EDA rings were incubated overnight with lipopolysaccharide (LPS), LPS+NS398 (selective COX-2 inhibitor) or NS398 alone. Vessel rings were then mounted in organ baths and relaxant responses to isoproterenol (ISOP) recorded on U44069-induced pre-contraction. Response to ISOP was further evaluated in either incubated or freshly isolated (e(-)) rings acutely exposed to NS398. Fresh and incubated (e(-)) EDAs were also analysed for COX-2 expression by Western blotting. LPS caused endothelium-dependent enhancement of β-adrenergic mediated relaxation. NS398 did not reverse endotoxin effects, suggesting that COX-2 did not have a mediating role. In the absence of LPS, NS398 significantly increased ISOP-induced relaxation. This finding, together with immunoblot detection of COX-2 in both fresh and incubated (e(-)) vessels, revealed the existence of a constitutive COX-2 exerting tonic inhibitory modulation on EDA β-adrenergic-mediated relaxation. The results support the possible role of endotoxin in the vascular disturbances associated with equine laminitis. Moreover, the involvement of COX-2 in the physiological regulation of EDA tone warrants further clinical investigation into the efficacy and safety of selective COX-2 inhibitors on digital circulation in horses.

Investigation of the metabolic activity of a bovine mammary epithelial cell line (BME-UV1)

The metabolic activity of a mammary epithelial cell line (BME-UV1) was evaluated on monolayers exposed, in serum free medium, to different concentrations (2-4-8 μM) of aflatoxin B1 (AFB1), a mycotoxin eliminated into milk especially as hydroxylated metabolite aflatoxin M1 (AFM1). After 4, 8, 12, 24 h of treatment, a dose and time dependent production of AFM1 has been detected. As the enzymes involved in the hydroxylation of AFB1 in bovine hepatocytes are mainly CYP1A and CYP3A, the results suggest that BME-UV1 express CYP450 isoenzymes which metabolize AFB1 thus representing a potential model for the investigation of the metabolic activity of bovine mammary epithelial tissue.