Toxic Effects of Furosine by Oral Intake on Liver and Kidney and Toxicokinetics Research in Mice Acute Toxicity Model

Abstract

In the acute toxicity model, 30 mice were randomly divided into 6 groups, control (without any treatment) and furosine-treated groups (4 h and 12 h group), by both orally administeration and tail vein injection. Furosine with the dosage of 0.24 g/kg body weight (b.w.) was administrated into the mice, and the liver and kidney tissue were dissected out. Organ index and biochemical indicators were obtained, and the concentration of furosine in liver and kidney tissue was quantified. Comparing with the control, furosine caused significant changes of biochemical indicators in liver and kidney tissue in 4 h and 12 h groups. This study for the first time provided evidences that furosine with the dosage of 0.24 g/kg posed acute adverse biological effects on the health of animals, and suggested that liver and kidney were the toxicity target organs.

Introduction

Maillard reaction is a key reaction in food processing courses, in which sugars and the degradation products attach to the residues of lysine and arginine, and amadori products named as Maillard reaction products (MRPs) are later produced (Henle, 2005). As one of the most famous member of MRPs, N(ε)-2-furoylmethyl-l-lysine (furosine, C₁₂H₁₈N₂O₄, Fig. 1) is a widely utilized indicator for the early and middle stage of Maillard reaction in processed food, whose structure is firstly identified in 1968 (Finot et al., 1968; Heyns, 1968). Furosine is commonly regarded as a marker of thermal treatment degree, protein loss and nutritional quality in foods (Ames, 1990; Mortier et al., 2000).

Fig. 1.

Chemical structure of furosine.

The concentration of furosine can be quantified in lots of food items, including formulas, milk, desserts, chocolate, cereals, honey and bakery sauce products (Rada-Mendoza et al., 2002; Villamiel et al., 2001), and its concentration is proved to positively related with storage time of food, temperature and time of food thermal process (Arnoldi et al., 2007; Rajchl et al., 2009; Seiquer et al., 2006; Troise et al., 2015). Though the technologies related with detection and quantification of furosine have been improved for several times, and the accuracy is stable, research related with the toxicological effects and toxicokinetics indicators of furosine is rare. In our recent study, furosine was validated to pose toxic effect on liver and kidney tissue in 35-day chronic toxicity model, embodied on the inhibition of body increase, pathological damage of liver and kidney tissue, etc., which might be related with cell inflammatory apoptosis induced by furosine (Li et al., 2018a). Furosine could also induced apoptosis of liver cancer cells through regulating transcription factor STAT1/2 and ubiquitination-related enzymes UBA7/UBE2L6 (Li et al., 2018b). However, the toxicokinetics characteristic and acute toxic effects of furosine with high dosages has not been evaluated. Thus, it is meaningful to investigate the toxicokinetics indicators of furosine in kidney and liver tissue of the mice administrated by gavage, as well as to elucidate the acute negative effects of furosine on animals and humans, aiming to determine the reference value of furosine in foods in further research. Here, we attempted to construct the acute toxicity model and measure the changing concentration of furosine in liver and kidney tissue and blood by high performance liquid chromatography (HPLC). Meanwhile, biochemical analysis (liver and kidney) were carried out to evaluate the acute toxicity of furosine.

Materials and Methods

Materials    Furosine was purchased from PolyPeptide (Strasbourg, France), whose purity was above 95 %. Elisa detection kits of TBil (total bilirubin), ALT (alanine transaminase), AST (aspartate transaminase), CR (creatinine), γ-GT (γ-glutamyl transferase), T-AOC (total antioxidant capacity), SOD (superoxide dismutase), MDA (malondialdehyde) and GSH (glutathione) were purchased from Jiancheng (Nanjing, China). Homogenate buffer (including proteases) and BCA quantification kit were purchased from Beyotime (China).

Animals feeding and acute toxicity model    ICR mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (license number SCXK 2012-0001 Beijing, China). Animals were kept in cages with a constant temperature of 25 °C and relative humidity of 50 %. The mice were acclimatized for 5 d before the formal experiment. All procedures for animal experimentation were performed according to China's guidelines for animal care, confirming to internationally accepted principles in the care and use of experimental animals (NIH publications No. 8023, in 1978). Animal experiments were approved by the Ethics Committee of Chinese Academy of Agriculture Sciences (Beijing, China) (Permission code: CAS 20171015; Permission date: 2017/10/15). Thirty female ICR mice (20 ± 2 g, 6-week old, 5 mice/group) were utilized in this study, and the acute toxicity test of furosine was performed as per the “Fixed Dose procedure” of OECD Test Guideline 420 (OECD, 2008). Furosine sample was dissolved in distilled water, and 0.2 mL was administrated by gavage to a mouse at the dose of 0.24 g/kg b.w.. The mice were fasted 2 h prior to dosing treatment, alive animals were sacrificed 4 h and 12 h after the treatment, and liver/kidney tissue was dissected out. Blood samples were gathered from femoral artery over time, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h and 12 h after the administration of furosine, then the serum samples were gathered by centrifugation of mice blood samples (3 000 rpm, 10 min). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Complete blood count (CBC)    Blood samples were placed into heparin-containing tube. The numbers of red and white blood cells and platelets were counted by DxH 800 Coulter machine (Beckman, USA).

Determination of furosine by UPLC MS/MS    For serum, triple volume of methanol (containing 0.1 % formic acid) was added into serum. After a full vortex and then placed in the 4 °C fridge for 1 hr, the tubes were centrifuged at 10 000 rpm×5 min, and the supernatant of each sample was collected for UPLC MS/MS analysis. For kidney and liver tissues, one hundred milligrams of kidney sample or liver sample were added into 1 mL tubes and incubated in 1 mL 50 % methanol for 7.5 min, until they had completely dissolved. The suspensions were then centrifuged at 12 000 rpm for 10 min. The upper layer (500 µL) was collected into new 1.5 mL glass tubes and 5 µL sample was analyzed using a UHPLC Q-Orbitrap, triplicate measurements in each aliquots.

UPLC condition (analytical parameters of liquid chromatography) was as follows, a waters column BEH C18 (130 Å, 1.7 µm, 2.1 mm X 50) was set at 40 °C, while the autosampler was maintained at 10 °C. The optimized chromatographic conditions were achieved at a flow rate of 0.4 mL/min with a mobile phase consisting of acetonitrile (mobile phase B) and 0.1 % formic acid aqueous solution (mobile phase A). The gradient elution process was: 0–2 min, 1 %–50 % B; 2–2.1 min, 50 %–1 % B; 2.1–3 min, 1 % B. The injection volume was 10 µL. And then, further MS/MS parameters were as follows, positive electrospray ionization was used for detection and the source parameters were selected as follows: spray voltage 4.0 kV, source temperature 150 °C, desolvation temperature 350 °C, desolvation gas flow of 400 L/h. The chromatographic profile was recorded in MRM mode and the characteristic transitions were monitored in order to improve selectivity using UPLC (ACQUITY UPLC I-Class) MS/MS (XEVO TQ-S) (Waters). MassLynx software (Version 4.1, Waters) was used for instrument control and data analysis. All relevant parameters are summarized in Table 1.

Table 1. The relevant parameters of UPLC MS/MS detection

Formula	MW	Molecular ion (m/z)	Fragment ion (m/z)	Cone(V)	Collision(V)	RT(min)
C12H18N2O4	254.286	255.26	83.81	76	20	0.34

Biochemical analysis    The organ tissue was cut into small pieces on ice, then homogenated by homogenate machine (ART Miccra, Germany), and the sample were gathered after centrifugation (3000 rpm, 10 min). The biochemical parameters ALT, AST and TBil were measured for liver tissue, CR and γ-GTP were measured for kidney, SOD, T-AOC, MDA and GSH were measured for both liver and kidney, according to the protocols of Elisa kits. The final optical density (OD) value was measured using microplate reader.

Statistical analysis    All data were expressed as mean ± standard deviation (SD) from several independent experiments (n=5). Statistical analyses were performed using the software SPSS 13.0 (SPSS Inc, USA). An analysis of variance (ANOVA) and Tukey test were used to determine the differences among the treatments. P value less than 0.05 were considered statistically significant (p < 0.05).

Results and Discussion

As a commonly applied early-stage indicator of Maillard reaction, furosine widely exists in heat-treated food products, especially in dairy products. Currently, the acute toxicity mice model was constructed to detect overall liver/kidney damage caused by furosine, as well as to investigate the metabolism of furosine in vivo.

Through quantification of furosine in blood, liver tissue and kidney tissue, we revealed toxicokinetics of furosine by UPLC-MS/MS analysis. Considering furosine to be taken from various foods, furosine was firstly administrated into mice by gavage to mimic the oral administration style in the present acute toxicity model. The results showed that the concentration of furosine in serum reached the peak value (926.6 ± 288.5 µg/L) at the time point of 0.5 h, then decreased gradually and was below 100 µg/L at 4 h. Through tail vein administration, the concentration of furosine decreased gradually after the injection (0.25 h), which was also below 100 µg/L at the time point of 4 h (Fig. 2A). The delay time (0.5 h) of the peak value by oral administration suggested that intake of furosine into blood from gastrointestinal tract required at least 0.5 h. By oral administration, both in kidney and liver tissue, the concentration of furosine reached the peak value at the time point of 4 h (90.3 ± 18.9 µg/kg in kidney, 40.1 ± 6.6 µg/kg in liver, respectively). Through tail vein administration, both in kidney and liver tissue, the concentrations of furosine also reached the highest values at the time point of 4 h (73.5 ± 16.1 µg/kg in kidney, 36.8 ± 6.0 µg/kg in liver, respectively). Being different from furosine detection in blood samples, high-dosage furosine still existed in kidney and liver 12 h after the administration, indicating that furosine migrated into the organs from blood, and kidney and liver were the toxicity target organs, which was in accordance with our previous results (Fig. 2B) (Li et al., 2018a). Results of liver/kidney analysis showed that the concentration of furosine reached the peak at 4 h, suggesting that furosine might enter into capillary vessels through intestinal absorption, then migrated into superior and inferior caval vein and arterial blood, later reached the target organs, i.e. liver and kidney. The whole course requires the further exploration and validation by fluorescence labeling or isotopic tracing in animal models, which deserves more attention and research.

Fig. 2.

Detection of furosine in mice serum, liver tissue and kidney tissue by HPLC-MS/MS, in oral intake model. A. Concentration of furosine in serum, when comparing with 0.25 h group, * p < 0.05; B. Concentration of furosine in liver tissue and kidney tissue, when comparing with the Control, * p < 0.05, when comparing with the one in 4 h group, ^# p < 0.05. All the data were represented as mean ± SD (n=5).

Through calculations of liver index and kidney index, we found that there was no significant difference of the organ index between the control mice and the treated mice (Fig. 3A). However, through measuring biochemical indicators in serum, liver tissue and kidney tissue, we found that liver and kidney might be the toxicity target organs. An administration of furosine caused expression changes of ALT, AST, CR, γ-GT, TBil, T-AOC, SOD and GSH, which reflected functions of liver and kidney, and antioxidation capacities. Comparing with the control level, expressions of ALT, AST, TBil in liver tissue were higher significantly (p < 0.05), CR and γ-GT in kidney tissue markedly increased (p < 0.05), T-AOC, SOD and GSH both in liver and in kidney expressed less than the control with statistical significance, and expression of MDA was markedly higher than the control (p < 0.05) (Fig. 3 B–G). Combined with the above results, furosine was proved to affect liver function and kidney function, as well as to induce oxidative stress in liver and kidney tissues.

Fig. 3.

Organ index and biochemical parameters for oral intake model. A. Liver index and kidney index; B–G. ALT, AST, TBil in liver tissue, CR, γ-GT and MDA in kidney tissue, T-AOC, SOD, GSH both in liver and in kidney. All data were represented as mean ± SD (n=5).

By CBC, we found that when comparing with the control level, the number of the total leukocyte count (WBC), lymphocyte (Lymph), neutrophil (Gran) and median cell (Mid) in 12 h group increased significantly (p < 0.05), the number of blood platelets (PLT) and red blood cell count (RBC) decreased significantly (p < 0.05). Moreover, there was significant difference of these indicators between in 4 h group and 12 h group (p < 0.05, Table 2).

Table 2. CBC of mice of mice blood in sub-acute toxicity model

Groups	WBC (109/L)	Lymph (%)	Mid (%)	Gran (%)	RBC (1012/L)	PLT (109/L)
Control	3.61±0.35	69.89±7.53	6.92±0.74	6.71±0.50	6.17±0.65	628.19±57.56
4 h group	3.62±0.44	74.35±11.55a	7.02±0.61a	7.33±1.06a	5.69±0.51a	580.57±89.42a
12 h group	4.84±0.75ab	79.19±13.46ab	7.52±1.23ab	9.03±1.83ab	5.17±0.77ab	421.64±90.55ab

Hematological data are represented as mean ± SD (n=5) and analyzed by Tukey test. a, p < 0.05 vs Control; b, p < 0.05 vs 4 h group.

The above results indicated that though furosine entered into liver and kidney within 4 h, it needed a longer time to accumulate, transfer and take adverse effects in liver/kidney tissue. In biochemical analysis, up-regulation of ALT, AST and TBil reflected damage caused by furosine in liver tissue (Hultcrantz et al., 1986; Li et al., 2010; Wasan et al., 2001), and up-regulation of CR and γ-GT indicated damage caused by furosine in kidney tissue (Levin and Cystatin, 2005), and up-regulation of MDA and down-regulation of SOD, T-AOC and GSH further verified that oxidative stress induced by furosine might lead to liver damage and renal damage, which could be elucidated by oxidative damages in other accumulating studies (Lozovoy et al., 2011; Barakat et al., 2018; Li et al., 2017; Loguercio et al., 2001; Marotta et al., 2009; Queisser et al., 2011). In CBC, several indicators were regarded as important parameters to reflect physiological changes in animal toxicological evaluation (Liang et al., 2009). In the present manuscript, the increase of WBC (10⁹/L), Lymph (%), Mid (%) and Gran (%), as well as the reduce of PLT (10⁹/L) and RBC (10¹²/L) suggested the function disorders, organism dystrophia and inflammatory reactions of mice treated by furosine, which might be achieved by adverse effects on hematopoietic function and immune system caused by 12 h influence of furosine (Ajuogu et al., 2015).

Conclusion

Our data validated that intake of high dosage furosine through oral administration or tail vein administration led to damages of kidney and liver, embodying on renal and hepatic functional biochemical indicators, pathological conditions, etc. Meanwhile, the metabolic and diffusing time of furosine from stomach or vein to organs was primarily determined, which provided valid evidences for toxicokinetics study of furosine or other harmful constituents in foods. These results may also improve our understanding of risk assessment of the components in foods and provide guidelines for further food safety evaluations of furosine.

Acknowledgments    We thank financial support from Fundamental Research Funds for the Central Non-profit Research Institution (Y2019PT06), the Ministry of Modern Agro-Industry Technology Research System of China (CARS-36), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS12).

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