Industrially produced trans-fatty acids are potent promoters of DNA damage-induced apoptosis

Abstract

trans-Fatty acids (TFAs) are unsaturated fatty acids harboring at least one carbon-carbon double bond in trans configuration, which are categorized into two groups according to their origin: industrial and ruminant TFAs, hereafter called iTFAs and rTFAs, respectively. Numerous epidemiological studies have shown a specific link of iTFAs to various diseases, such as cardiovascular and neurodegenerative diseases. However, there is little evidence for underlying mechanisms that can explain the specific toxicity of iTFAs, and how to mitigate their toxicity. Herein, we show that iTFAs, including elaidic acid (EA) and linoelaidic acid, but not rTFAs, facilitate apoptosis induced by doxorubicin (Dox), triggering DNA double-strand breaks. We previously established that EA promotes Dox-induced apoptosis by accelerating c-Jun N-terminal kinase (JNK) activation through mitochondrial reactive oxygen species (ROS) overproduction. Consistently, iTFAs specifically enhanced Dox-induced JNK activation. Furthermore, Dox-induced pro-apoptotic signaling by iTFAs was blocked in the presence of oleic acid (OA), the geometrical cis isomer of EA. These results demonstrate that iTFAs specifically exert their toxicity during DNA damage-induced apoptosis, which could be effectively suppressed by OA. Our study provides evidence for understanding the difference in toxic actions between TFA species, and for new strategies to prevent and combat TFA-related diseases.

INTRODUCTION

trans-Fatty Acids (TFAs) are a type of unsaturated fatty acid (UFA) characterized by the presence of one or more trans carbon-carbon double bonds. In the human body, enzymes responsible for fatty acid desaturation introduce exclusively cis double bonds into fatty acids (Oteng and Kersten, 2020). Consequently, enzymatically synthesized UFAs are hereafter referred to as cis-fatty acids (CFAs), containing only cis double bonds, while TFAs are exclusively derived from dietary sources (Micha and Mozaffarian, 2008). These TFAs are categorized into two groups based on their origin: industrial TFAs (iTFAs) and ruminant TFAs (rTFAs). iTFAs, such as elaidic acid (EA, C18:1 t9) and linoelaidic acid (LEA, C18:2 t9,t12), are produced as byproducts during food manufacturing processes like the partial hydrogenation of edible oils containing CFAs, and are commonly found in processed foods, including snacks and fast food items (Gebauer et al., 2007). By contrast, rTFAs, such as trans-vaccenic acid (tVA, C18:1 t11), rumenic acid (RA, C18:2 c9,t11) and palmitelaidic acid (PEA, C16:1 t9), are primarily generated through bacterial biohydrogenation of CFAs in the rumen of ruminant animals, including cows and sheep, and are abundant in meat and dairy products. Compelling epidemiological evidence has shown that the consumption of TFAs, especially iTFAs, is associated with an increased risk of various health disorders, including cardiovascular diseases (CVDs), systemic inflammation, metabolic syndrome, and neurodegenerative diseases (NDs) (Mensink et al., 2003; Morris et al., 2003; Micha and Mozaffarian, 2009; Bendsen et al., 2011; Lopez-Garcia et al., 2005). These findings are supported by studies conducted on mouse models (Morris et al., 2006; Bassett et al., 2009; Dorfman et al., 2009). However, the structure-toxicity relationships of TFAs are still unclear, since the molecular mechanisms of how TFAs exert their toxicity have remained unknown.

We have established toxicity mechanisms of TFAs in response to extracellular ATP (eATP), one of the damage-associated molecular patterns (DAMPs) released from injured cells (Hirata et al., 2017b; Hirata et al., 2020b), and DNA damage (Hirata et al., 2020a; Hirata et al., 2021). eATP triggers apoptosis by activating the apoptosis signal-regulating kinase 1 (ASK1)-p38 MAP kinase pathway (Noguchi et al., 2008). This apoptosis signaling begins with the generation of reactive oxygen species (ROS) downstream of the P2X purinoceptor 7 (P2X₇), mediated by NADPH oxidases, and ROS in turn activates ASK1, whose activation is tightly regulated by various interacting molecules (Hirata et al., 2017a; Hirata, 2019). We demonstrated that TFAs, particularly EA, enhance eATP-induced activation of the ASK1-p38 pathway, thereby promoting apoptosis in both macrophages and microglial cells (Hirata et al., 2017b; Hirata et al., 2020b). On the other hand, TFAs have a pro-apoptotic role when cells are exposed to DNA damage associated with double-strand breaks (DSBs) induced by doxorubicin (Dox), a DNA topoisomerase II inhibitor (Hirata et al., 2020a). While p53, a renowned tumor suppressor, is typically activated in response to DNA damage, regulating key genes like p21, Bcl-2-associated X protein (Bax), and p53 upregulated modulator of apoptosis (Puma) in the DNA damage response (Hafner et al., 2019), we found that TFAs promote apoptosis independently of p53; instead, they rely on the mitochondrial c-Jun N-terminal kinase (JNK)-Sab-ROS axis (Hirata et al., 2020a). Sab, an adaptor protein of JNK localizing on the mitochondrial outer membrane, recruits activated JNK in response to various stresses, including DNA damage (Win et al., 2018). This leads to increased mitochondrial ROS (mitoROS) production and JNK activation, resulting in hyperactivation of JNK, and ultimately apoptosis as a positive feedback mechanism (Hirata et al., 2020a). Taking into account that eATP and DNA damage are closely associated with the pathogenesis of TFA-related diseases, including CVDs and NDs (Burnstock, 2017; Sluyter, 2017; Jackson and Bartek, 2009), our findings shed light on the underlying mechanisms of these diseases (Hirata, 2021). Furthermore, our very recent study revealed the specific toxicity of iTFAs during eATP-induced apoptosis. We demonstrated that iTFAs, including EA and LEA, effectively enhance the activation of ASK1 and p38, leading to cell death induced by eATP. In contrast, rTFAs, including tVA, RA, and PEA, had minimal effect on eATP-induced cell death, aligning with prior epidemiological studies that linked iTFAs to various disorders, including CVDs (Mensink et al., 2003; Morris et al., 2003; Micha and Mozaffarian, 2009; Bendsen et al., 2011; Lopez-Garcia et al., 2005). Moreover, we explored the potential beneficial effects of all-cis polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5 c5,c8,c11,c14,c17) and docosahexaenoic acid (DHA, C22:6 c4,c7,c10,c13,c16,c19), and found that co-administration of PUFAs effectively counteracted the pro-apoptotic effects of iTFAs by blocking ASK1 hyperactivation. These results underscored the distinct pro-apoptotic role of iTFAs during eATP stimulation and highlighted PUFAs as potent mitigators of TFA toxicity. However, it remains unknown whether iTFAs also exert specific toxicity upon DNA damage, and whether their toxicity could be ameliorated by PUFA treatment.

We here report that iTFAs (i.e. EA and LEA) potently promote Dox-induced activation of JNK, and ultimately cell death, whereas rTFAs (i.e. tVA, RA and PEA) hardly do. In addition, we found that the pro-apoptotic effect of EA during DNA damage was strongly inhibited by co-treatment with oleic acid (OA, C18:1 c9) as a cis isomer of EA, but not that with PUFAs. These results indicate that iTFAs have a particularly strong pro-apoptotic activity during DNA damage, which could be mitigated in the presence of OA, providing insight into a better understanding of the TFA toxicity, and developing new strategies for prevention and treatment of TFA-related diseases.

MATERIALS AND METHODS

Cell culture

U2OS cells were cultured in Dulbecco’s Modified Eagle Medium (Nacalai Tesque, Kyoto, Japan), containing 10% heat-inactivated fetal bovine serum (Sigma, Burlington, MA, USA) and 1% penicillin-streptomycin solution (Nacalai Tesque) in 5% CO₂ at 37°C. Doxorubicin was purchased from Sigma.

Preparation and treatment of fatty acids

OA (Nacalai Tesque), EA (Sigma), linoleic acid (LA, C18:1 c9,c12), LEA, RA, palmitoleic acid (POA, C16:1 c9), PEA, arachidonic acid (AA, C20:4 c5,c8,c11,c14), EPA and DHA (Cayman, Ann Arbor, MI, USA), and tVA (Olbracht Serdary Research Laboratories, Toronto, ON, Canada) were prepared as described previously (Hirata et al., 2020a). Briefly, fatty acids were dissolved in 0.1 N NaOH at 70°C, and then conjugated with fatty acid-free BSA (pH 7.4: Wako, Tokyo, Japan) at 55°C for 10 min to make 5 mM BSA-conjugated fatty acid stock solutions containing 10% BSA. Cells were treated with various concentrations of BSA-conjugated fatty acids by diluting stock solutions in a medium without fetal bovine serum (final BSA concentration was set to 1%).

Immunoblot analysis

Cells were lysed in ice-cold lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton-X100, 10% Glycerol, and 1% protease and phosphatase inhibitor cocktail (Nacalai Tesque). After centrifugation, the cell extracts were resolved by SDS-PAGE, and were analyzed as described previously (Yamada et al., 2023). The antibodies used for immunoblotting were against phospho-JNK, JNK, caspase-3 (Cell Signaling, Danvers, MA, USA), β-actin (Santa Cruz, Dallas, TX, USA). The blots were developed with ECL (Merck Millipore, Burlington, MA, USA), and detected with ChemiDoc Touch Imaging System (BioRad, Hercules, CA, USA).

Cell viability assay

Cell viability was assayed as described previously (Sekiguchi et al., 2019). U2OS cells were seeded on 96-well plates (10,000 cells/well). After indicated stimulation, cell viability was determined using Cell Titer 96 Cell Proliferation Assay (Promega, Madison, WI, USA), according to the manufacturer's protocol. The absorbance was read at 490 nm using a microplate reader (iMark microplate reader, BioRad). Data are normalized to control without stimulus, unless noted otherwise.

Lipid analysis

Lipids were extracted by the method of Bligh and Dyer (Bligh and Dyer, 1959). Isolated lipids were methylated with 2.5% H₂SO₄ in methanol, and the resulting fatty acid methyl esters were then extracted with hexane. Gas chromatography tandem mass spectrometry (GC-MS/MS) analysis was performed with a GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan) equipped with Zebron ZB-FAME 60 m x 0.25 mm x 0.20 µm (Phenomenex, Torrance, CA, USA), and data were analyzed as described previously (Hirata et al., 2023).

Statistics

All the values are expressed as means ± SD, and statistical analyses were performed using GraphPad Prism software (v.9.3.0). All experiments were repeated at least three independent times, and statistical significance was determined with the one-way ANOVA analysis followed by Tukey-Kramer test.

RESULTS

iTFAs specifically promote Dox-induced cell death

Before assessment of the toxicological activities of the five major food-derived TFAs (Fig. 1), we first checked the cytotoxicity of these TFAs in human osteosarcoma U2OS cells that were previously used to establish the toxicity mechanisms of TFAs in response to DNA damage (Hirata et al., 2020a; Hirata et al., 2021). As shown in Fig. 2A, single treatments of the respective TFAs did not significantly affect cell viability, indicating that treatment with these TFAs alone is not cytotoxic. We next performed a toxicological analysis of the five TFAs. U2OS cells were pretreated with these TFAs, treated with 0.5 µg/mL Dox, and assayed for cell viability. We found that pretreatment with iTFAs (EA and LEA) substantially reduced cell viability, while that with rTFAs (tVA, RA, and PEA) did not (Fig. 2B). Consistently, Dox-induced caspase-3 activation (cleavage), a hallmark of apoptosis, was drastically enhanced in the presence of iTFAs (Fig. 2C). In addition, pretreatment with representative 16- and 18-carbon CFAs, POA, and OA/LA, respectively, did not diminish cell viability upon Dox treatment (Fig. 2D). Notably, we previously demonstrated that one of the typical saturated fatty acids, palmitic acid (C16:0), did not have a pro-apoptotic effect on Dox-induced apoptosis (Hirata et al., 2020a). Altogether, these data suggest that iTFAs harbor a unique pro-apoptotic activity in response to Dox-induced DSBs among diverse fatty acids. Of note, by GC-MS/MS analysis, we confirmed that all the rTFAs were incorporated to almost the same amount as EA (Fig. 3), which indicates that the observed difference in cell viability was not due to decreased uptake of rTFAs.

Fig. 1

Structures of the fatty acids used in this study. The figures indicate the locations of carbon-carbon double bonds. In parentheses, the details are indicated as follows: abbreviation, the count of carbon atoms, and the specifics, including the number, configuration (t for trans, c for cis), and positions of the carbon-carbon double bonds.

Fig. 2

iTFAs specifically promote Dox-induced apoptosis. (A) U2OS cells were treated with the indicated TFAs for 16 hr at the indicated concentrations, and assayed for cell viability. Data are shown as mean ± SD (n=3). NS: not significant (vs control without fatty acid). (B) U2OS cells were pretreated with the indicated TFAs for 12 hr at the indicated concentrations, stimulated with Dox at 0.5 µg/mL for 24 hr, and assayed for cell viability. (C) U2OS cells were pretreated with the indicated TFAs for 12 hr at 200 µM, treated with Dox at 0.75 µg/mL for 8 hr, and then subjected to immunoblotting with the indicated antibodies. (D) U2OS cells were pretreated with the indicated CFAs for 12 hr at the indicated concentrations, stimulated with Dox at 0.5 µg/mL for 24 hr, and assayed for cell viability. Data are shown as relative cell viability (mean ± SD, n=3), normalized first with the viability of cells treated with the respective fatty acid at the same concentrations, and secondly with that of Dox-stimulated cells without fatty acid. NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 (vs Dox-stimulated cells without fatty acid).

Fig. 3

Comparison of the intracellular amount of exogenously added TFAs. U2OS cells were treated with the indicated FAs for 12 hr at 50 µM and 200 µM. Intracellular TFAs were extracted, methylated, and subjected to GC-MS/MS analysis. Relative amounts of incorporated TFAs are shown as mean ± SD (n=3). NS, not significant; *, p<0.05 (vs EA).

iTFAs trigger JNK hyperactivation upon Dox treatment

We previously showed that in the presence of EA, Dox-induced JNK activation and mitoROS generation were enhanced in a positive feedback manner, thereby promoting apoptosis (Hirata et al., 2020a). We therefore assessed the effects of the five TFAs on JNK activation in response to Dox. As shown in Fig. 4A, pretreatment with EA clearly increased JNK activation upon Dox treatment, while that of tVA had an intermediate effect on JNK activation. Considering that there was no significant effect of tVA on Dox-induced cell death (Fig. 2B), this slight increase in JNK activity appears to be unrelated to apoptosis. More importantly, pretreatment with LEA apparently elevated Dox-induced phosphorylation of JNK, whereas that with either RA or PEA did not significantly increase it (Fig. 4B-C), in agreement with their effects on cell viability during Dox treatment (Fig. 2B). These results suggest that iTFAs trigger Dox-induced hyperactivation of JNK possibly through the JNK-Sab-ROS axis, leading to promotion of apoptosis.

Fig. 4

iTFAs trigger hyperactivation of JNK in response to Dox. (A-C) U2OS cells were pretreated with the indicated FAs for 12 hr at 200 µM, treated with Dox at 0.5 µg/mL for the indicated periods, and then subjected to immunoblotting with the indicated antibodies.

OA counteracts pro-apoptotic action of EA during DNA damage

Our very recent study demonstrated that PUFAs, including AA, EPA, and DHA, but not OA and LA that have one or two cis carbon-carbon double bonds, potently suppress eATP-induced apoptosis by targeting ASK1 to block its hyperactivation (Hirata et al., 2023). However, interestingly, we found that neither of the PUFAs nor LA reversed the pro-apoptotic effect of EA upon Dox treatment, except that DHA only slightly prevented it (Fig. 5A). On the other hand, OA strongly suppressed it in a dose-dependent manner (Fig. 5A-B). In line with this result, co-treatment with DHA scarcely reduced JNK hyperactivation in response to Dox, but that with OA clearly suppressed it (Fig. 5C-D). Of note, OA co-treatment slightly but significantly reduced the amount of EA incorporated into cells only when both fatty acids were treated at 200 µM (Fig. 5E-F). Collectively, these data suggest that pro-apoptotic action of EA during DNA damage can be antagonized by co-treatment of OA, rather than that of PUFAs, contrary to the case of eATP-induced apoptosis (Hirata et al., 2023).

Fig. 5

OA counteracts pro-apoptotic action of EA upon Dox treatment. (A, B) U2OS cells were pretreated with 200 µM EA and 200 µM CFAs (A) or OA at the indicated concentrations (B) for 12 hr, stimulated with 0.5 µg/mL Dox for 24 hr, and assayed for cell viability. Data are shown as relative cell viability (mean ± SD, n=3), normalized with the viability of cells stimulated with Dox without fatty acid. *, p<0.05; **, p<0.01; ***, p<0.001 (A: vs Dox-stimulated cells with EA only). (C, D) U2OS cells were pretreated with 200 µM EA and 100 µM DHA or OA for 12 hr, treated with 0.5 µg/mL Dox for the indicated time periods, and then subjected to immunoblotting with the indicated antibodies. (E, F) U2OS cells were treated with EA and OA for 12 hr at 0, 50, or 200 µM. Cellular lipids was extracted, methylated, and subjected to GC-MS/MS analysis. Relative amount of incorporated EA is shown as mean ± SD (n=3). NS: not significant; *, p<0.05 (vs 0 µM OA).

DISCUSSION

In this study, we demonstrated that EA and LEA, the primary TFAs found in industrially produced foods (namely, iTFAs), effectively facilitated Dox-induced apoptosis signaling (Fig. 2, 4). On the other hand, tVA, RA, and PEA, major TFAs present in ruminant foods (namely, rTFAs), did not exhibit a similar pro-apoptotic effect (Fig. 2, 4). Moreover, the representative CFAs, POA, OA and LA, the geometrical cis isomers of PEA, EA and LEA, respectively, did not show pro-apoptotic properties (Fig. 2D). Taken together, these data suggest that iTFAs possess a distinctive ability to promote DNA damage-induced apoptosis, which may explain their specific association with TFA-related diseases, such as CVDs and NDs (Mori et al., 2015; Dawczynski and Lorkowski, 2016), as illustrated in Fig. 6. The differences in the positions of carbon-carbon double bonds between iTFAs and rTFAs lead to speculation that the n-9 trans double bond, common between EA and LEA, is essential for their pro-apoptotic action. Importantly, the specificity of the toxic actions between TFA species is common between eATP- and DNA damage-induced apoptosis (Hirata et al., 2023), implicating a common toxic mechanism of action shared particularly among iTFAs, despite the difference in their toxicity mechanisms according to the stress conditions. Notably, EA, which enhanced Dox-induced apoptosis to a similar extent as LEA (Fig. 2B), is more abundant in the diet compared to di-ene TFAs like LEA (Food Safety Commission in Cabinet Office, 2012). Indeed, EA (~10 µM) is much more abundant in the human blood in comparison to LEA (~2 µM), suggesting that EA plays the most significant role in TFA-related diseases among TFA species. It should be noted that although, to date, there has been no reliable evidence showing how much TFAs are contained in human tissues, TFA levels in tissues are estimated to be much higher than those in human blood, based on the observations from mouse studies (Hirata et al., 2017b; Dorfman et al., 2009; Hussein et al., 2007; Liu et al., 2010; Larqué et al., 2000), and the range of TFA concentrations (50-200 µM), where toxicity was significantly observed in this study, would be physiologically relevant, as discussed in detail in our previous study (Hirata et al., 2023).

Fig. 6

The schematic of the toxic actions of TFAs and the protective actions of OA. Upon Dox stimulation, iTFAs, including EA and LEA, enhanced JNK activation and apoptosis (solid arrow), while rTFAs, including tVA, RA, and PEA, did not (dotted gray arrow). OA reversed pro-apoptotic activity of EA, which could be utilized for the prevention and therapeutics for TFA-related diseases, including CVDs and NDs.

We further showed that OA, but not PUFAs, possesses a protective effect on TFA toxicity during DNA damage (Fig. 5). Based on the structural similarity between EA and OA (i.e. geometrical isomers), OA might be able to mitigate the pro-apoptotic effect of EA by competing with EA for being incorporated into the phospholipids in the mitochondrial membrane, the presumable site where EA exerts its toxicity upon DNA damage (Hirata et al., 2020a). It would also be plausible that cellular uptake of EA could be partially blocked by OA (Fig. 5E-F). In contrast, in the case of eATP-induced apoptosis, the pro-apoptotic activity of iTFAs was canceled by co-treatment with PUFAs, but not that with OA (Hirata et al., 2023). We demonstrated that PUFAs target ASK1 and reverse its hyperactivation induced by eATP (Hirata et al., 2023), while the target of EA is an upstream kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (Hirata et al., 2017b); thus, it is unlikely that PUFAs play a protective role by directly competing with the target of EA. Also, a possible reason why OA cannot compete with EA during eATP-induced apoptosis is that the mode of existence in which EA exerts its pro-apoptotic effect may be unique to EA. EA might be incorporated into specific phospholipid species into which OA is not normally incorporated, and thereby enhancing CaMKII activation in response to eATP. These possibilities should be explored in future research. Intriguingly, we previously demonstrated that EA promoted apoptosis induced by cisplatin, a DNA alkylating agent, by a different molecular mechanism from Dox (a DNA DSB-inducing agent)-induced apoptosis (Hirata et al., 2021). In a future study, we would like to investigate whether iTFAs also specifically promote apoptosis induced by other types of DNA damage, such as DNA alkylation, and which CFAs could counteract the toxic actions of TFAs.

Notably, a randomized crossover trial in 1990 demonstrated that the consumption of a diet enriched with EA is associated with higher low-density lipoprotein (LDL)-cholesterol levels and lower high-density lipoprotein (HDL)-cholesterol levels in comparison with that of a diet enriched with OA (Mensink and Katan, 1990), proposing a pro-atherogenic role of EA through the dysregulation of cholesterol metabolism. This finding has been supported by later epidemiological and mouse studies (Oteng and Kersten, 2020). By contrast, monounsaturated fatty acids (MUFAs), including OA, and PUFAs have been characterized as anti-atherogenic FAs, whose consumption has been associated with reduced risk of CVDs (Livingstone et al., 2012). Based on the above-mentioned assumptions and evidence, it can be speculated that consumption of foods rich in PUFA and OA might be a promising means of preventing and alleviating TFA-related diseases, not just by normalizing cholesterol metabolism, but also by ameliorating excessive cell death at the lesion areas. Future studies may provide more evidence to support a more comprehensive understanding of TFA toxicity, leading to the development of novel prevention and therapeutic approaches using MUFAs and PUFAs.

ACKNOWLEDGMENTS

We thank all members of Lab of Health Chemistry for helpful discussions. This work was supported by JSPS/MEXT KAKENHI Grant Numbers JP20K07011, JP20KK0361, JP23K06111 (Y.H.), JP21H02620 and JP21H00268 (A.M.), and by MHLW Grant Number JPMH23KA3004 (Y.H.). This work was also supported by the Mitsubishi Foundation, the Japan Foundation of Applied Enzymology, the Uehara Memorial Foundation, the Takeda Science Foundation, the Japan Foundation for Aging and Health, Sapporo Bioscience Foundation, and Lotte Research Promotion Grant.

Conflict of interest

The authors declare that there is no conflict of interest.

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