Effects of leptin Arg25Cys on peripheral mononuclear cell counts and antibody response to vaccination in beef cattle.

A single nucleotide polymorphism (SNP) in the leptin gene that results in Arg25Cys has been associated with beef carcass quality and milk composition in dairy cattle. However, leptin (LEP) also plays a role in immune performance and hence it was important to determine whether selection based on this SNP would negatively affect immune cell numbers or antibody production. LEP c.73C>T was assessed for effects on immune cell counts and antibody titres in 27 beef cattle herds (n = 556). A commercial rabies vaccine had been administered to these animals. Prior to being vaccinated, counts of several important mononuclear cells (total and activated B lymphocytes, total and activated T helper and T cytotoxic, WC1 T lymphocytes and monocytes) as well as baseline serum antibody titres were determined for each animal. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and mononuclear cell types were again counted. Counts of six different cell types were significantly associated with the LEP genotype; however, no consistent patterns were observed between LEP genotype (TT, CT or CC) and peripheral blood mononuclear cell populations. Significant differences in the production of rabies antibodies in response to vaccination were observed relative to LEP genotype. Our results suggest that selection for either the C or T allele would not detrimentally impact on the measured indicators of immune function in beef cattle.

Effects of aflatoxin B(1) in a liver cell line from rainbow trout (Oncorhynchus mykiss).

The cytotoxic response to aflatoxin B(1) (AFB) was investigated in RTL-W1, a cell line derived from the normal liver of a mature rainbow trout (Oncorhynchus mykiss). AFB altered RTL-W1 morphology and ultrastructure and inhibited DNA synthesis. The effective concentration required for 50% inhibition (EC(50)) of DNA synthesis, after 2 days of treatment was 0.04 mug/ml. This response was compared with that of two other salmonid, but non-liver, cell lines [rainbow trout gonad (RTG-2) and Chinook salmon embryo (CHSE-214)]. Although RTG-2 cells were as sensitive as RTL-W1 cells (EC(50) for inhibition of DNA synthesis was 0.05 mug/ml), CHSE-214 cells were unresponsive to AFB at concentrations as high as 2 mug/ml. After a single AFB exposure, RTL-W1 sublines were isolated that had phenotypic changes typical of malignant transformation. These were increased growth rate, reduced contact inhibition of growth, altered cellular morphology and growth in soft agar. In addition, RTL-W1 metabolized AFB: the major metabolites, aflatoxin (AFL) and aflatoxin M(1) (AFM), were detected by thin-layer chromatography and HPLC. The relative amounts of these metabolites, unlike those observed with RTG-2 cells, were in close agreement with those produced by trout liver in vivo. Thus, RTL-W1 could provide a sensitive in vitro model system for studying the action of biotransformation requiring xenobiotics. Overall, the observed responses were similar to those reported for liver cells in AFB-exposed trout, suggesting that RTL-W1 cells are suitable for studying cytotoxic effects and malignant transformation in vitro.

Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity.

A cell line, RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.