Safety Evaluation of Branched-Chain Amino Acids in Centropomus Viridis Brain Cells: In Vitro Assay.

Resumen

Branched-chain amino acids (BCAAs), composed of L-

leucine, L-isoleucine, and L-valine, are essential amino acids

in the nutrition of various animal species, including marine

fish. Traditionally considered precursors of protein synthesis,

BCAAs also act as key signaling molecules and metabolic

modulators. Although its physiological role has been widely

studied, information on its toxicity and safety, especially in

fish cell systems, remains limited. The species Centropomus

viridis, due to its high dietary requirements of amino acids,

represents a relevant model to explore the effects of BCAAs

on brain cells in an in vitro environment. Immortal cultures

of Centropomus viridis brain cells were established from

juvenile specimens, following standardized protocols of

enzymatic disintegration and seeding in 96-well plates. The

cells were kept in L-15 medium supplemented with 10% fetal

bovine serum and antibiotics, incubated at 25 °C without

CO₂. The treatments consisted of exposure to BCAAs

solutions (L-leucine, L-isoleucine, and L-valine) at

concentrations of 50, 100, 250, 500, 1000, 2500, 5000 and

1

0000 μg/mL, instantly prepared from sterile stocks.

Negative controls (cells without BCAAs) and positive

controls were included. Cell viability was assessed using the

MTT assay, adding 10μL of MTT solution (5 mg/mL) per

well and measuring absorbance at 570 nm after 4 hours of

incubation. Additionally, trypan blue exclusion was

performed by incubating the cells with the dye for 5 minutes

and counting viable and non-viable cells under light

microscopy. The evaluation criteria included percentage of

viability with respect to the negative control and observation

of cell morphology. BCAA treatments even at concentrations

of 1000, 2500, 5000 and 10000 μg/mL did not produce

significant alterations in cell viability, with percentages

greater than 95% compared to the negative control. No

morphological changes or increase in trypan blue exclusion

ABSTRACT

were observed. At the maximum concentration evaluated

(5000 and 10000 μg/mL), no decrease in viability was

detected (up to 95% of live cells), without reaching criteria

of significant cytotoxicity or evident morphological damage.

The positive control showed a reduction in viability of less

than 60%, validating the sensitivity of the experimental

system. Therefore, the cellular safety limit for BCAAs in this

line is above 5000 and 10000 μg/mL, with no appreciable

adverse effects above this threshold. The results obtained

coincide with the few previous reports in mammals, where

BCAAs present low toxicity even at high concentrations. The

absence of relevant cytotoxic effects in brain cells of

Centropomus viridis suggests a wide tolerance to these amino

acids, in accordance with the physiological needs of marine

species with high protein requirements. However, the

literature indicates a lack of controlled studies in humans and

other species, which makes it difficult to extrapolate these

data to broader clinical and dietary contexts. This study

demonstrated that branched-chain amino acids are harmless

and safe for brain cells of Centropomus viridis under in vitro

conditions, even at elevated concentrations of up to 10000

μg/mL. The methodology used, based on MTT assays and

exclusion of trypan blue, provides robust criteria for the

toxicological evaluation of nutritional supplements in marine

fishes. These findings contribute to knowledge about food

safety in aquaculture and highlight the need to expand

research to other cell systems and species, as well as to

generate comprehensive metabolic and toxicological data to

establish accurate recommendations on the use of BCAAs in

animal and human feed.