A search for acrolein scavengers among food components

Brain stroke is a major cause of being bedridden for elderly people, and preventing stroke is important for maintaining quality of life (QOL). Acrolein is a highly reactive aldehyde and causes tissue damage during stroke. Decreasing acrolein toxicity ameliorates tissue injury during brain stroke. In this study, we tried to identify food components which decrease acrolein toxicity. We found that 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine decreased acrolein toxicity. These compounds neutralized acrolein by direct interaction. However, the interaction between acrolein and taurine was not so strong. Approximately 30 mM taurine was necessary to interact with 10 μM acrolein, and 2 g/kg taurine was necessary to decrease the size of mouse brain infarction. Taurine also slightly increased polyamine contents, which are involved in decrease in the acrolein toxicity. Mitochondrial potential damage by acrolein was also protected by taurine. Our results indicate that daily intake of foods containing 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine may prevent severe injury in brain stroke and improve the quality of life for elderly people.


Introduction
Brain stroke is a severe symptom caused by a blockage of brain blood vessels and the main cause of being bedridden for elderly people. Stroke is also a leading cause of dementia and depression (Johnson et al. 2016). Approximately 15 million people suffer a brain stroke each year worldwide (http:// www. emro. who. int/ health-topics/ stroke-cereb rovas cular-accid ent/ index. html). As stroke incidence and severity increase during aging (Nakayama et al. 1994;Uemura et al. 2016), this disease is one of the primary reasons for disability among the elderly. Since the number of people of advanced age is increasing globally, finding a novel method to prevent and treat brain stroke is important.
We have reported that acrolein (CH 2 = CH-CHO), an unsaturated aldehyde, is strongly involved in tissue damage in brain stroke ). Compared to reactive oxygen species (ROS), such as hydrogen peroxide, that exerts cell growth inhibition at 0.2-0.4 mM, acrolein is much more toxic as it inhibits cell growth at several µM level (Sharmin et al. 2001;Yoshida et al. 2009). Acrolein is produced mainly by oxidation of the polyamine spermine in vivo (Pegg 2013), easily reacts with proteins, and acrolein-conjugated proteins contribute to cytotoxicity. Acrolein inactivates GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Nakamura et al. 2013), and disrupts the cytoskeleton by conjugating with tubulin (Uemura et al. 2019), vimentin and actin (Uemura et al. 2020). Acrolein is also involved in the tissue damage of Sjögren's syndrome patients by activating matrix metalloproteinase-9 (Uemura et al. 2017). Related to metabolic syndrome, acrolein-modified low-density lipoprotein can be taken up by macrophages via scavenger receptor class A type 1 (SR-A1), resulting in foam cell formation (Kobayashi et al. 2021;Watanabe et al. 2013). Administration of acrolein scavenger N-acetylcysteine and its derivatives decreased the size of brain infarction in a mouse model by increasing the stability of glutathione S-transferases that are involved in acrolein detoxification . This suggests that effective acrolein scavengers can be a preventive agent for brain stroke. Accordingly, we looked for food components which can detoxify acrolein toxicity.

Cell culture
Mouse neuroblastoma-derived Neuro2a cells were cultured in Dulbecco's Minimal Essential Medium (MEM) supplemented with 50 U/ml streptomycin, 100 U/ml penicillin G and 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO 2 . For cell growth analysis, cells were inoculated at : None : Compound : 10 μM Acrolein : Compound + 10 μM Acrolein Fig. 1 Effect of food components on acrolein toxicity. Neuro2a cells were cultured with 10 µM 2-furanmethanethiol (2-FMT), 30 µM cysteine methyl and ethyl ester (CysMe and CysEt), 100 µM alliin, 10 mM lysine, or 30 mM taurine, respectively, in the presence or absence of 10 µM acrolein, and growth of cells was followed by counting live cells. The data are presented as mean ± SD of triplicate determinations. The structures of food components are shown together with the graphs 2 × 10 5 cells/well to 6-well plates (BD Falcon, the size of growing area is approximately 10 cm 2 /well) and cultured for 12 h. Then, 10 µM acrolein (Tokyo Chemical Industry, Tokyo, Japan), and 2-furanmethanethiol (Tokyo Chemical Industry), cysteine methyl ester and ethyl ester (Tokyo Chemical Industry), alliin (Sigma-Aldrich), lysine (Nacalai Tesque) and taurine (Nacalai Tesque) at the indicated concentrations, were added to the medium, and cell growth was followed for 3 days. The viability of cells was monitored by staining with 0.2% trypan blue solution.

Measurements of glutathione, taurine, polyamine and protein contents
Glutathione and taurine contents in cells were determined using a Glutathione Assay Kit (Cayman Chemical) and a Taurine Assay Kit (Cell Biolabs), respectively. Cells were washed three times with ice-cold phosphate buffered saline (PBS), homogenized in MES buffer containing 0.4 M 2-(N-morpholino)ethanesulphonic acid, 0.1 M phosphate and 2 mM EDTA, pH 6.0, and mixed with an equal volume of 0.2 M trichloroacetic acid at 4 °C for 10 min. After centrifugation, supernatants were diluted with MES buffer and total glutathione and taurine contents were measured according to the manufacturer's protocol. Polyamine contents in cells were determined as described previously (Igarashi et al. 1986). Protein contents in cells were determined with a BCA protein assay kit (Nacalai Tesque) using bovine serum albumin as a standard.

Photochemically induced thrombosis (PIT) model mice
All animal experiments were approved by the Institutional Animal Care and Use Committee of Chiba University and carried out according to the Guidelines for Animal Research of Chiba University. PIT model mice were prepared using 8-week-old male C57BL/6 mice (22-26 g) as described previously (Saiki et al. 2009). Taurine was administered intraperitoneally, just before the induction of infarction. The Effect of taurine on brain infarction (A) and changes of taurine content in Neuro2a cells in the presence or absence of acrolein and taurine (B). A Experiments were performed as described in Materials and methods using 9 control mice and 10 taurine-injected mice. **p < 0.01. B Neuro2a cells were incubated with 30 mM taurine for 2 days, and then incubated with or without 10 μM acrolein for another 2 days, and taurine content in the cells was measured. The data are presented as mean ± SD of triplicate determinations. *p < 0.05; **p < 0.01

Fig. 5
Effect of food components on polyamine contents. Neuro2a cells were cultured with the food components, and polyamine contents were measured as described in Materials and methods in the presence (black bar) and absence (white bar) of 10 µM acrolein. Data are shown as means ± SD of triplicate determinations. ***p < 0.005 efficacy of taurine was calculated as the volume of decreased infarction per 2 g administered per kg of mouse body weight.
No mouse presented signs of paresis, convulsion, remarkable weight loss or any other symptoms.

Measurement of the level of free acrolein in the presence of anti-acrolein reagents
Free acrolein was measured according to the method of Alarcon (Alarcon 1968). The reaction mixture (0.3 mL) containing 67 mM Na + -phosphate buffer, pH 7.5, 10 μM acrolein, and reagents at indicated concentrations was incubated at 37 ºC for 2.5, 5, 10 and 20 min. At each interval, 50 μL of the reaction mixture was taken out and mixed with an equal volume of solution containing 92 mM m-aminophenol, 172 mM hydroxylamine hydrochloride and 3 M HCl. After the mixture was boiled for 10 min, acrolein content was determined by HPLC according to the method of Bohnenstengel et al. (1997), using 10 μL supernatant after centrifugation. Fluorescence of 7-hydroxyquinoline (an acrolein derivative) was measured at an excitation wavelength of 358 nm and an emission wavelength of 510 nm.

Staining of mitochondria
Neuro2a cells were cultured with or without 10 µM acrolein and/or 30 mM taurine for 3 days. Cells were then washed with fresh medium and incubated with 0.5 µM MitoTracker Orange (Invitrogen) and 1 μg/ml Hoechst 33,342 (Sigma-Aldrich) for 30 min at 37 ºC in a fresh medium. Fluorescence was observed under an Evos FL Auto2 fluorescence microscope with Celleste image analysis software (Invitrogen). All images were captured in the same settings of microscope and any cropping or adjustment was applied.

Statistical analysis
Statistical analysis was performed as described previously (Uemura et al. 2019). For comparison of multiple groups, one-way ANOVA followed by Bonferroni's multiple comparisons test was used.

Food components which decreased acrolein toxicity
Acrolein is a strongly toxic compound for cells and attacks cysteine, lysine and histidine residues in proteins to form protein-conjugated acrolein (PC-Acro) ). Since a thiol group reacts preferentially with acrolein (Danyal et al. 2016), we sought food components containing a sulfur atom as acrolein scavengers. Mouse neuroblastoma derived Neuro2a cells were incubated with acrolein and several compounds to assess cytotoxicity. As shown in Fig. 1, 10 µM acrolein inhibited cell growth. Addition of 10 µM 2-furanmethanethiol (2-FMT), a flavor component of coffee, 30 µM cysteine methyl and ethyl esters (CysMe and CysEt), contained in green onions, together with acrolein recovered cell growth. Alliin (100 µM), a garlic component, lysine (10 mM), high in red meat and beans, and taurine (30 mM), which is rich in squids and clams, also diminished the inhibitory effect of acrolein on cell growth.

Acrolein conjugation activity of food derived compounds
The ability of compounds to neutralize acrolein was then analyzed by acrolein conjugation assay. Acrolein and compounds were incubated together and the remaining free acrolein was measured by HPLC. As shown in Fig. 2, 10 μM 2-FMT, 30 μM CysMe and CysEt, and 100 μM alliin rapidly neutralized free acrolein. Lysine (10 mM) and taurine (30 mM) also are able to conjugate acrolein, although a high concentration was necessary. This suggests that taurine may reduce acrolein toxicity via a different mechanism together with the direct elimination of acrolein.

Recovery of glutathione contents by acrolein scavengers
Glutathione is a natural detoxifier for acrolein in tissue (Tomitori et al. 2012). Thus, the effect of compounds on the level of glutathione was examined. As shown in Fig. 3, although acrolein treatment significantly decreased glutathione content, addition of acrolein scavengers recovered glutathione to normal levels. These results suggest that the compounds interact with acrolein and disturb acrolein conjugation with glutathione, which was observed in N-acetylcysteine administrated into mouse brain after induction of infarction ).

Effect of taurine on brain infarction and cell growth of acrolein-treated Neuro2a cells
It was then determined whether taurine changes the size of brain infarction in mice, because acrolein scavengers N-acetylcysteine and its derivatives decreased the size of brain infarction . As shown in Fig. 4A, addition of 2 g/kg taurine significantly decreased the size of brain infarction. How the taurine content changes in the presence of acrolein in Neuro2a cells was investigated. As shown in Fig. 4B, the addition of 10 μM acrolein slightly increased the content of taurine. If the intracellular water space of Neu-ro2a cells was similar to rat liver cells [5.5 μl cell volume/ mg protein (Watanabe et al. 1991)], taurine concentration increased approximately from 10 to 15 mM by addition of 10 μM acrolein. However, inhibition of cell growth was not recovered. When 30 mM taurine was added, intracellular taurine concentration increased to approximately 30 mM, at which concentration acrolein was detoxified (see Fig. 1). The results indicate that more than 30 mM taurine are necessary to detoxify acrolein toxicity.

Effect of acrolein and its scavengers on polyamine contents
We next studied how acrolein scavengers influence polyamine content in cells. Polyamine content did not change significantly by the addition of acrolein scavengers except taurine (Fig. 5). Taurine significantly increased both putrescine and spermidine contents, but spermine content was not affected.
Polyamines possess two primary amino groups that potentially react with acrolein (Tsutsui et al. 2014). To test whether polyamines can neutralize free acrolein, an acrolein conjugation assay was performed. As shown in Fig. 6A, putrescine, spermidine and spermine interacted with acrolein. These results suggest that the increase in polyamine content caused by taurine may contribute to acrolein detoxification through interaction with free acrolein.
The levels of polyamine biosynthetic and degrading enzymes were then measured to confirm the effect of taurine on polyamine contents. As shown in Fig. 6B, ornithine decarboxylase (ODC), a rate limiting enzyme for polyamine biosynthesis (Pegg 2006), was slightly decreased in acrolein-treated cells and increased in taurine-treated cells. Taurine treatment counteracted the decrease in ODC level by acrolein. S-adenosylmethionine decarboxylase 1 (AMD1), another rate-limiting enzyme for polyamine biosynthesis (Lim et al. 2018), was significantly decreased by acrolein, and taurine slightly recovered the decrease. Spermidine synthase (SPDS) was not affected by either acrolein or taurine treatment. Spermine synthase (SPMS) decreased greatly by acrolein treatment, but taurine slightly recovered the level of SPMS. Spermine oxidase (SMO), which catalyzes spermine degradation to produce acrolein, was increased slightly by acrolein. Another polyamine degradation pathway consisting of acetylpolyamine oxidase (AcPAO) and spermidine/ spermine N 1 -acetyltransferase (SSAT) (Battaglia et al. 2014) was not affected by acrolein and taurine treatment. These results suggest that the increase in putrescine and spermidine levels in taurine-treated cells may be partially involved in the detoxification of acrolein by taurine.

Protective effect of taurine on mitochondrial damage by acrolein
There are reports that acrolein targets mitochondrial potential and induces cellular oxidative stress (Mohammad et al. 2012;Sun et al. 2006;Wu et al. 2020). It is also reported that taurine is identified as a constituent of mitochondrial tRNAs (Suzuki et al. 2002), and is known to be important for mitochondrial function (Jong et al. 2021). As shown in Fig. 6 Neutralization of acrolein by polyamines (A) and effect of acrolein and taurine on the levels of polyamine biosynthetic and metabolic enzymes (B). A Acrolein (10 µM) was incubated with 10 mM each of putrescine, spermidine or spermine, respectively, for 2, 5, 10 and 20 min and remaining free acrolein was measured as described in Materials and methods. Data are represented as mean ± SD of triplicate determinations. B Neuro2a cells were cultured with or without 10 µM acrolein and/or 30 mM taurine for 3 days. Levels of proteins were measured by Western blotting and the bands were quantified as described in Materials and methods, and expressed as mean ± SD of triplicate determinations. The levels of polyamine biosynthetic enzymes (ODC, AMD1 and SPMS), but not metabolic enzymes (SMO, Ac-PAO and SSAT), were changed significantly by acrolein or taurine. *p < 0.05; **p < 0.01; ***p < 0.005 against none ◂ Fig. 7, 20 µM acrolein diminished mitochondrial potential. When cells were treated with 30 mM taurine, the mitochondrial potential was retained after acrolein exposure. These results indicate that taurine protects mitochondria from acrolein damage.

Discussion
In this study, we sought food components which reduce acrolein toxicity to identify novel anti-stroke compounds. Our previous work demonstrated that cysteine derivatives ameliorated tissue injury of brain stroke in a mouse model ). Thus, we tested the acrolein detoxification effect of sulfur containing compounds. Thiol compounds, i.e. 2-FMT and cysteine methyl and ethyl esters counteracted acrolein toxicity at 10-30 µM (Fig. 1). These compounds neutralized free acrolein in less than 20 s (Fig. 2), and prevented the decrease of glutathione by acrolein (Fig. 3). The results indicate that 2-FMT and cysteine methyl and ethyl esters neutralize free acrolein and rescued cells from acrolein toxicity. We suggest that taking foods and drink containing these components such as green onion and coffee can offer a benefit for preventing brain stroke.
We also found that sulfur-containing compounds alliin and taurine, and the amino acid lysine decreased acrolein toxicity. Alliin and lysine neutralized free acrolein even slower compared to 2-FMT and cysteine methyl and ethyl esters, but this ability to neutralize acrolein probably contributes to the acrolein detoxification. Interestingly, high concentrations of taurine rescued cells from acrolein toxicity. Taurine not only neutralizes acrolein weakly (Fig. 2), but also increases putrescine and spermidine levels (Fig. 5). These polyamines also neutralized acrolein (Fig. 6), suggesting that the increase in polyamine content is also involved in the decrease in acrolein toxicity by taurine. Western blot analysis revealed that ODC1 and AMD1, both of which are rate-limiting enzymes in polyamine synthesis (Pegg 2016), are upregulated by taurine in the presence of acrolein (Fig. 6). Acrolein also Fig. 7 Effect of acrolein and taurine on mitochondrial potential. Neuro2a cells were cultured with or without 10 µM acrolein and/ or 30 mM taurine for 3 days. Cells were then washed and incubated with Mitotracker Orange and Hoechst 33,342 for 30 min at 37 ºC in a fresh medium, and fluorescence was observed as described in Materials and methods. Red; mitochondria, Blue; nuclei. Experiments were repeated three times and reproducible results were obtained diminished mitochondrial potential (Fig. 7), but taurine protected the decrease in the mitochondrial potential from acrolein. These results indicate that taurine decreases acrolein toxicity due to direct interaction with acrolein as well as through the increases in polyamine contents and mitochondrial functions. Taurine (2 g/kg) could decrease significantly the size of brain infarction (Fig. 4). Recently, it has been reported that taurine chloramine (0.5 mg/kg) protects against postischemic brain injury (Seol et al. 2021). However, this may be due to the presence of chloramine together with taurine.
Brain stroke is one of the major causes of the decrease in quality of life (QOL) in the elderly (Johnson et al. 2016). Our results suggest that daily intake of foods and drink containing thiol compounds (2-FMT, CysMe and CysEt), alliin, lysine and taurine, such as coffee, garlic, green onions, beans, squid and clams is probably useful to prevent severe injury in brain stroke and to improve QOL.