In‐Vitro Toxicological and Proteomic Analysis of Furan Fatty Acids Which are Oxidative Metabolites of Conjugated Linoleic Acids

Abstract

Furan fatty acids (furan-FA) are oxidative products of conjugated linoleic acids (CLA) and may therefore be ingested when CLA-containing food or food-additives are consumed. Due to the presence of a furan ring structure the question arises whether furan-FA may have toxic properties on enterocytes and liver cells. Here we show that furan-FA neither have toxic effects in human colon cancer cell line Caco-2 nor in human hepatoma cell line HepG2 at concentrations that could be relevant for humans. At concentrations up to 100 μM, all tested furan-FA isomers showed no pronounced cytotoxicity and did not affect cellular proliferation or apoptosis up to concentrations of 500 μM. In addition, furan-FA was neither genotoxic in the micronucleus test using Chinese hamster lung fibroblasts (V79) nor in the Ames test independent of the presence or absence of rat liver homogenate for enzymatic activation of the furan ring structure. A proteomic approach revealed that 48 proteins were differentially expressed when Caco-2 cells were incubated with up to 1 mM of 10,13-epoxy-10,12-octadecadienoic acid (10,12-furan-FA). Three of the 30 proteins that could be identified by MALDI-TOF analysis were upregulated and were associated with lipid droplet biogenesis. The remaining 27 proteins were downregulated and were considered to be associated with general cellular processes such as DNA replication and transcription, protein biosynthesis and protein processing, lipid and energy metabolism. From the proteomic data we conclude that furan-FA is predominantly stored in lipid droplets thereby downregulating cellular metabolic activity and driving the cells into a state of rest.

Effect of docosahexaenoic acid and furan fatty acids on cytokinesis block micronucleus cytome assay biomarkers in astrocytoma cell lines under conditions of oxidative stress

“…Physiological concentrations of FFA as determined from whole blood of healthy individuals was estimated to be <1 mg/mL, with the most abundant FFA isomer, F 6 around 32.3 6 8.5 ng/mL . The supraphysiological levels of FFA used in our study were, however, comparable with those used in previous studies [Lengler et al, 2012] and exerted significant differences between cell line responses, but only in regards to cellular proliferation (NDI), necrosis, and the frequencies of BN with NPBs ( Fig. 3).…”

“…In contrast to U118MG, U87MG cell proliferation increased and followed an "inverted U-shaped" trend with the greatest rates observed at 1 mg/mL (1.9 to 3.1 mM) and 10 mg/ mL (19.0 to 31.0 mM). The decrease in cell proliferation in U118MG cells by FFA also correlated with recent toxicology studies, which demonstrated that FFA downregulated genes associated with general cellular processes such as DNA replication, transcription, translation, and protein biosynthesis [Lengler et al, 2012] and may have therefore induced a state of cell cycle arrest within the U118MG astrocytes. When challenged with an acute dose of H 2 O 2 , DNA damage levels (BN with MNi and BN with NPBs) in U118MG cells increased in the presence of FFA supplementation at 1,000 mg/mL, indicating a likelihood of misrepair of DNA strand breaks leading to the formation of acentric chromosome fragments and dicentric chromosomes from which MNi and NPBs originate.…”

“…When challenged with an acute dose of H 2 O 2 , DNA damage levels (BN with MNi and BN with NPBs) in U118MG cells increased in the presence of FFA supplementation at 1,000 µg/mL, indicating a likelihood of misrepair of DNA strand breaks leading to the formation of acentric chromosome fragments and dicentric chromosomes from which MNi and NPBs originate. Lengler et al [] also reported that the 8,10‐FFA, 9,11‐FFA, 10,12‐FFA, and 11,13‐FFA isomers significantly decreased cell viability at 500 µM within 48 hr in enterocytes and hepatocytes, whereas FFA concentrations below 100 µM were not cytotoxic. Despite emerging evidence of FFA having potent antioxidant capacities, we could not find any protective effect of FFA against H 2 O 2 ‐induced cytotoxicity or genotoxicity, indicating a lack of benefit of FFA regarding elements of cell viability.…”

TPD52 expression increases neutral lipid storage within cultured cells

“…In support of this hypothesis, another TPD52-like protein TPD52L2 has been reproducibly identified within lipid droplet fractions (Hodges and Wu, 2010) from oleic-acid-treated A431 cells stably expressing stomatin (Umlauf et al, 2004), Hep39 cells (Sato et al, 2006), U937 cells (Wan et al, 2007), differentiated 3T3-L1 cells (Cho et al, 2007) and 3T3-L1 cells cultured to stimulate lipolysis (Brasaemle et al, 2004). Increased TPD52L2 levels have also been reported in response to furan fatty acid treatment of Caco-2 cells (Lengler et al, 2012), and in the first reported mouse knockout model for any Tpd52-like gene, smaller body length and absent or minimal hepatic lipidosis were reported in Tpd52l2 −/− females (Adissu et al, 2014). Like TPD52, TPD52L2 isoforms are predicted to lack 14-3-3-binding sites in most tissues (Boutros et al, 2003).…”

Potential favourable health effects of some dietary uncommon fatty acids

“…Several in vitro studies support this hypothesis (Graff et al, 1984;Okada et al, 1996;Fuchs and Spiteller, 1999) as well as studies conducted in humans (Wahl et al, 1994;Zheng et al, 2016;Tovar et al, 2017). Moreover, in vitro FuFAs modulate lipid metabolism in adipose tissues (Lengler et al, 2012;Lauvai et al, 2019). The 3-carboxy-4methyl-5-propyl-2-furanpropanoic acid (CMPF), a degradation product of FuFAs, also derived from the metabolism of n-3 PUFAs, could prevent or even reverse hepatic steatosis (Prentice et al, 2018;Dai et al, 2019;Mohan et al, 2019).…”