PMC

Mouse primary hepatocytes cultured in 0.5 ml Dulbecco's modified Eagle's medium were co-incubated with different numbers of (A) eggs or (B) ESP. After 72 h, alanine tranaminase (ALT), a biomarker for hepatotoxicity, was measured. A combination of 5 mM D-galactosamine hydrochloride (D-gal) and 1 µg/ml rTNF-α (D-gal/TNF-α) was used as a known hepatotoxic control. As a negative control, hepatocytes were incubated with PBS. Data are displayed as the mean ± S.D. from two experiments each performed in duplicate.

Hepatocytes were co-incubated with 10 µg/ml ESP or SEA, or with ESP or SEA first depleted of both omega-1 and IPSE with a mixture of anti-IPSE and anti-omega-1 monoclonal antibodies (each 5 µg/ml). After 72 h, the presence of ALT in the medium was measured. A combination of 5 mM D-galactosamine hydrochloride (D-gal) and 1 µg/ml rTNF-α (D-gal/TNF-α) was employed as a known hepatotoxic control and negative control cultures used PBS. Depletion of both IPSE and omega-1 diminished the toxicity of ESP and SEA by 60% and 58%, respectively. Data are displayed as the mean ± S.D. from two experiments each in duplicate. * P<0.05 for values significantly different from ESP and SEA is based on paired analysis (one sided paired Student's t-test).

(A) Hepatocytes were co-incubated with various amounts of rIPSE. After 72 h, the presence of ALT in the medium was measured. As positive controls, cells were co-incubated with 10 µg/ml each of ESP or SEA. A combination of 5 mM D-galactosamine hydrochloride (D-gal) and 1 µg/ml rTNF-α (D-gal/TNF-α) was employed as a known hepatotoxic control and negative control cultures used PBS. (B) Pre-incubation of a specific monoclonal antibody (5 µg/ml) with rIPSE abolished the latter's toxicity and respectively decreased cytotoxicity of ESP and SEA by 32 and 35%. Data are displayed as the mean ± S.D. from two experiments each performed in duplicate. *P<0.05, **P<0.001 and ***P<0.0001 using a one-sided paired Student's t-test. The hatched line represents the control baseline.

ESP was fractionated by Mono Q anion exchange chromatography. The fraction not bound to the column (flow-through (‘FT’)) and each subsequent eluted fraction were incubated with mouse primary hepatocytes in 0.5 ml Dulbecco's modified Eagle's medium. After 72 h, ALT, a biomarker for hepatotoxicity, was measured. Fraction #11 induced significant liver cell injury *P<0.05 compared to negative control cultures employing PBS. Other fractions (6–10, 12–17) had some hepatotoxic effect. No hepatotoxic effect was present in fractions 1–5. Total ESP (10 µg/ml) and D-gal/TNF-α (5 mM/1 ug/ml) were included as positive controls. Data are displayed as the mean ± S.D. from two experiments each performed in duplicate.

(A) Hepatocyte cultures (0.5 ml in Dulbecco's modified Eagle's medium were co-incubated with various amounts of purified native omega-1, and egg-derived material (10 µg/ml). After 72 h, ALT, a biomarker for hepatotoxicity, was measured. Polymyxin B (80 µg/ml) was included in co-incubations with omega-1 to neutralize any potential LPS. Control cultures contained polymyxin B alone. Aspergillus oryzae T2 RNAse (25 U/ml) was also used as a comparison in light of omega-1's described T2 RNase activity. A combination of 5 mM D-galactosamine hydrochloride (D-gal) and 1 µg/ml rTNF-α (D-gal/TNF-α) was employed as a known hepatotoxic control and negative control cultures used PBS. (B) Pre-incubation of a specific monoclonal antibody (5 µg/ml) with omega-1 abolished the latter's toxicity and respectively decreased cytotoxicity of ESP and SEA by 47 and 33%. Data are presented as the means ± SD from two independent experiments each performed in duplicate. *P<0.02, **P<0.001 and ***P<0.0001 using a one-sided paired Student's t-test.