Investigating the effects of thymol and cinnamaldehyde in T-2 toxin-treated hepatic cell cultures of chicken origin

Abstract

Trichothecenes are among the most significant mycotoxins, causing serious agricultural problems worldwide. One representative of these is T-2 toxin, which is often found in poultry feed. Due to its harmful effects, it is important to investigate substances that can counteract it. Such compounds include thymol and cinnamaldehyde, whose anti-inflammatory and antioxidant activities are well-documented. In this study, the effects of 24 h exposure to T-2 toxin (100 nM), thymol, and cinnamaldehyde (10 and 100 μg/mL in both cases) and their combinations with the toxin were examined in chicken-derived 3D liver cell cultures. Cellular viability was assessed by measuring the lactate dehydrogenase (LDH) activity. Inflammatory parameters were monitored by Luminex method, while the levels of oxidative and autophagy markers were determined by chicken-specific ELISA tests. Our results indicated that neither compound significantly affected cellular viability. T-2 toxin decreased interferon-γ (IFN-γ) and “Regulated on Activation, Normal T cell Expressed and Secreted” (RANTES), while increased macrophage colony-stimulating factor (M-CSF). Sole treatment of cinnamaldehyde reduced interleukin (IL)-10, IFN-γ, and RANTES, whereas thymol elevated IL-6 but lowered M-CSF and RANTES. In combination treatments, cinnamaldehyde further decreased IFN-γ, M-CSF, and RANTES, while thymol increased IL-6, IL-10, IFN-γ, and M-CSF relative to the T-2 toxin-treated cells. Thymol reduced nuclear factor (erythroid-derived 2)-like 2 (NRF-2), whereas cinnamaldehyde increased NRF-2 during combination treatment compared to the T-2 toxin-treated cells. Malondialdehyde (MDA) levels were lowered by cinnamaldehyde. Both compounds increased microtubule-associated protein 1 light chain 3 beta (LC3B) in combination treatments; however, cinnamaldehyde reduced P62 compared to the T-2 toxin-treated group. These results show that T-2 toxin exerted its negative effects by influencing inflammatory processes, but it was not cytotoxic. Thymol and cinnamaldehyde exhibited anti-inflammatory and, in some cases, even pro-inflammatory activity. In addition, both protective substances helped to initiate autophagy mechanisms in cells and influenced redox-related markers; cinnamaldehyde decreased MDA levels, while thymol modulated NRF-2 expression.