Lack of Acute Toxicity and Mutagenicity from Recombinant Epinephelus lanceolatus Piscidin Expressed in Pichia pastoris

Abstract

The antimicrobial peptide (AMP) piscidin was identified from Epinephelus lanceolatus and demonstrated to possess antimicrobial and immune-related functions. Supplementation of feed with recombinant Epinephelus lanceolatus piscidin (rEP)-expressing yeast pellets may minimize the excessive use of antibiotics and control pathogens in aquaculture or animal husbandry. However, before implementing rEP as a supplement, it is necessary to understand whether it harbors any toxicity. Since toxicological information on the topic is scarce, the present investigation was carried out to test whether rEP exhibits allergenic and/or toxic effects. In an oral acute toxicity test (OECD 425), Sprague Dawley (SD) rats were administered rEP dissolved in reverse osmosis water, yielding an LD₅₀ > 5000 mg/kg (no observed animal death). The compound was therefore classified as non-toxic by oral administration. In an acute respiratory toxicity test (OECD 403), heads and noses of SD rats were exposed to liquid aerosol for 4 h (the highest concentration that could be administered without causing any animal death), and a lethal concentration (LC₅₀) > 0.88 mg/L was obtained. The mass medium aerodynamics diameter (MMAD) of rEP aerosol particles was 8.18 μm and mass medium aerodynamics diameter (GSD) was 3.04, which meant that 25.90% could enter the airway (<4 μm) of a rat, and 58.06% (<10 μm) could be inhaled by humans. An ocular irritation test (OECD 405) with rEP powder was performed on New Zealand White (NZW) rabbits. Signs of irritation included conjunctival swelling and diffuse flushing 1 h after administration. The signs were less apparent after 24 h and disappeared after 72 h. The classification assigned to the powder was mild eye irritation. Skin sensitization was performed for a local lymphoproliferative test (OECD 442B) using BALB/c mice, with the highest soluble concentration of the rEP considered to be 100% test substance; formulations were diluted to 50% and 25%, and bromodeoxyuridine (BrdU) incorporation was used to measure the degree of lymphocyte proliferation. The stimulation indexes (SIs) were 1.06 (100%), 0.44 (50%), and 0.77 (25%), all of which were less than the cutoff value for a positive sensitization result (1.6). Negative response was also seen in the bacterial reverse mutation test (OECD 471), and no chromosomal effects on Chinese hamster ovary (CHO)-K1 cells were observed (OECD 487). Based on these six toxicity tests, rEP showed neither acute toxic effects in experimental animals nor mutagenicity. Thus, rEP can be considered safe for use in subsequent research on its application as a feed additive for poultry, cattle, or aquatic animals.

Keywords: Epinephelus lanceolatus piscidin; allergic effects; antimicrobial peptide; mutagenetic toxicity; recombinant piscidin; toxic effects.

Figure 1

Recovery from rEP-mediated mild eye irritation after 24 h. Observation of eye irritation in an individual rabbit treated with rEP at 1, 24, 48, and 72 h post-treatment. Images were captured with a hand-held retinal camera.