Effects of dichlorvos and formalin on fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells in primary culture

The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-14C]Stearic (18:0), [1-14C]linoleic (18:2n-6) and [1-14C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of phosphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial Δ9, Δ6 and Δ5 fatty acyl desaturase activities. Although, as expected, the cells were more active towards [1-14C]18:3n-3, the cells were unusually active towards [1-14C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit Δ9 and Δ6 desaturase. Changes in the distribution of radioactivity between individual phospholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor in fish and considering the importance of eicosanoids in the biochemistry of skin ,it is suggested that these agents may have direct effects on fish skin that may have important consequences for fish health in general

The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinisation of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-14C]18:2n-6 and [1-14C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolised by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while Δ6 desaturase activity was increased significantly with cell age, Δ5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures

Metabolism of 18:4n-3 (stearidonic acid) and 20:4n-3 in salmonid cells in culture and inhibition of the production of prostaglandin F2alpha (PGF2alpha) from 20:4n-6 (arachidonic acid)

Arachidonic acid (AA; 20:4n-6) is the precursor of a range of highly biologically active derivatives, collectively termed eicosanoids, including prostaglandins, thromboxanes, leukotrienes and lipoxins, that act as autocrine hormones regulating many physiological processes including haemostasis, reproduction, immune and inflammatory responses. Eicosapentaenoic (EPA; 20:5n-3) and dihomo-γ-linolenic (20:3n-6) acids modulate eicosanoid metabolism by both inhibiting the conversion of AA to eicosanoids whilst simultaneously being converted to eicosanoids with different, often attenuated, properties compared to their AA homologues. Eicosatetraenoic acid (20:4n-3) is a naturally occurring C20 polyunsaturated fatty acid (PUFA), present in fish oil at levels of around 1-2%, that has been suggested to be the active metabolite responsible for the anti-inflammatory effects of plant oils containing stearidonic acid (18:4n-3). However, the biochemical properties of 20:4n-3 in terms of cellular biology have rarely been investigated, partly due to difficulties in obtaining the fatty acid in high purity. In this paper, we describe methods for the medium scale laboratory preparation of high purity 20:4n-3, and investigate its metabolism in fish cell culture systems which normally contain significant amounts of n-3 PUFA. Thus the incorporation and metabolism of 18:4n-3 and 20:4n-3, and their distribution in phospholipid classes was studied in an established cell line from Atlantic salmon (Salmo salar) (AS), and the effects of 20:4n-3 on eicosanoid production studied in freshly isolated macrophages from rainbow trout (Oncorhynchus mykiss). Both 18:4n-3 and 20:4n-3 were preferentially esterified into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in contrast with the accumulation of AA in phosphatidylinositol. Incorporated 18:4n-3 was readily converted to 20:4n-3, and both fatty acids were further desaturated and elongated to EPA and 22:5n-3 but not 22:6n-3. Supplementation with 20:4n-3 decreased the conversion of AA into prostaglandins, as demonstrated by the decreased levels of PGF2α produced in trout macrophages supplemented with 20:4n-3 and AA compared to cells supplemented with AA alone. In addition, 20:4n-3 was converted into eicosanoids in fish cells as indicated by the presence of Δ17,18 12-HETE, Δ17,18 PGE1 and Δ17,18 PGF1α in extracts from rainbow trout macrophages incubated with 20:4n-3

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n-3), to eicosapentaenoic acid, 20:5(n-3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C18 to C20 polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n-3), and its elongation product, 20:4(n-3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 uM), U-14C (1 uM) or deuterated (d5; 25 uM) fatty acids. Stearidonic acid, 18:4(n-3), was metabolised to 20:5(n-3) in both cells lines, but more so in AS than in TF cells. Delta-5 desaturation was more active in TF cells than in AS cells, whereas C18 to C20 elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n-3)) were produced by both cell lines, although there was significant production of 22:5(n-3) in both cultures, especially when 20:4(n-3) was supplemented. We conclude that limited elongation of C18 to C20 fatty acids rather than limited fatty acyl Delta-5 desaturation accounts for the limited rate of conversion of 18:3(n-3) to 20:5(n-3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C18 to C20 PUFA by the turbot and the Atlantic salmon in vivo

Fatty acid metabolism in marine fish: Low activity of fatty acyl Δ5 desaturation in gilthead sea bream ( Sparus aurata ) cells

Marine fish are known to have an absolute dietary requirement for C20 and C22 highly unsaturated fatty acids. Previous studies using cultured cell lines indicated that underlying this requirement in marine fish was either a deficiency in fatty acyl Δ5 desaturase or C18-20 elongase activity. Recently, Ghioni et al. (Biochim. Biophys. Acta, 1437, 170-181, 1999) presented evidence that in turbot cells there was low activity of C18-20 elongase whereas Δ5 desaturase had high activity. In the present study, the fatty acid desaturase/elongase pathway was investigated in a cell line (SAF-1) from another carnivorous marine fish, sea bream. The metabolic conversions of a range of radiolabelled polyunsaturated fatty acids that comprised the direct substrates for Δ6 desaturase ([1-14C]18:2n-6 and [1-14C]18:3n-3), C18-20 elongase ([U-14C]18:4n-3), Δ5 desaturase ([1-14C]20:3n-6 and [U-14C]20:4n-3) and C20-22 elongase ([1-14C]20:4n-6 and [1-14C]20:5n-3) were utilized. The results showed that fatty acyl Δ6 desaturase in SAF-1 cells was highly active and there was substantial C18-20 elongase and C20-22 elongase activities. A deficiency in the desaturation/elongation pathway was clearly identified at the level of the fatty acyl Δ5 desaturase which was very low, particularly with 20:4n-3 as substrate. In comparison, the apparent activities of Δ6 desaturase, C18-20 elongase and C20-22 elongase were approximately 94-fold, 27-fold and 16-fold greater than that for Δ5 desaturase towards their respective n-3 polyunsaturated fatty acid substrates. The evidence obtained in the SAF-1 cell line is consistent with the dietary requirement for C20 and C22 highly unsaturated fatty acids in the marine fish, the sea bream, being primarily due to a deficiency in fatty acid Δ5 desaturase activity

Biocidi in preparati topici ed integrita' della berriera cutanea

Dottorato di ricerca in microbiologia del farmaco e del cosmetico. 8. ciclo. A.a. 1994-95. Coordinatore F. Negretti. Docente guida A. BetteroConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal