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Isopsoralen induces different subchronic toxicities and metabolomic outcomes between male and female Wistar rats
Regulatory Toxicology and Pharmacology 103 (2019) 1–9
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Isopsoralen induces different subchronic toxicities and metabolomic outcomes between male and female Wistar rats
T
Yue Zhanga,b,c,1, Xiao-mei Yuana,1, Yue-fei Wanga,b, Miao-miao Jianga,b, Ya-nan Bia, Ying Liud, Wei-ling Pua, Lei Songa,c, Ju-yang Huanga,c, Li-kang Suna,b, Zhi-xing Zhoue, Kun Zhoua,b,c,∗ a
Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin, 300193, China c Ministry of Education Key Laboratory of Traditional Chinese Medical Formulae, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China d College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, 010018, China e Tianjin Institute of Pharmaceutical Research, Tianjin, 300193, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Wistar rats Fructus psoraleae Isopsoralen Subchronic toxicity Metabolomics
Isopsoralen is a major active and quality-control component of Fructus Psoraleae, but lacks a full safety evaluation. We evaluated the oral toxicity of isopsoralen in Wistar rats treated for 3 months at doses of 0, 3.5, 7.0, and 14 mg/kg. Additionally, the plasma metabolomics of isopsoralen in male and female rats treated for 3 months at doses of 0 and 14 mg/kg were investigated by gas chromatography-mass spectrometry. Many abnormalities were observed in the isopsoralen-treated rats, including suppression of body weight gain, and changes in serum biochemical parameters and visceral coefficients. Histopathological changes in liver, pancreatic, and reproductive system tissues were also observed in the isopsoralen-treated rats. The metabolomic analyses showed alterations in many metabolites (19 in female rats; 28 in male rats) after isopsoralen administration. The significant changes in these metabolites revealed metabolomic alterations in the isopsoralentreated rats, especially in amino acid metabolism regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Furthermore, fatty acid metabolism comprised the main affected pathways in female rats, while lipid metabolism and energy metabolism were the main affected pathways in male rats.
1. Introduction Fructus Psoraleae, the seed of Psoralea corylifolia L., has been used extensively in China for its various biological activities, including antioxidant activity (Haraguchi et al., 2002; Tang et al., 2004), antimicrobial activity (Khatune et al., 2004; Citarasu et al., 2003), osteoblastic activity (Lim et al., 2009; Tsai et al., 2007), and estrogen-like effect (Zhang et al., 2005). Some studies have revealed the hepatotoxicity and cytotoxicity of Fructus Psoraleae in recent years (Nam et al., 2005; Cheung et al., 2009)(Mar et al., 2001). Isopsoralen is one of the two quality-control components of Fructus Psoraleae according to the Chinese Pharmacopoeia (2015 edition). The pharmacological activities of isopsoralen have been widely researched. One study showed
that isopsoralen had a protective effect against oxidative damage in human lens epithelial cells (Feng et al., 2014). Another study suggested that isopsoralen significantly inhibited the proliferation of sensitive and multidrug-resistant cancer cells, and had anticancer activity in vitro (Wang et al., 2011). Isopsoralen also exhibited osteoprotective effects in ovariectomized and orchidectomized mice (Yuan et al., 2016). However, few studies on the toxicity or safety evaluations of isopsoralen have been reported. One exception was a report that ingestion of isopsoralen for 28 days may influence metabolism and excretion in mice (Wang et al., 2012b #46), but there are no reliable predictive and prognostic biomarkers to assess the hepatotoxicity induced by isopsoralen. Clearly, not only the therapeutic effect, but also the safety of a drug is important, and thus a subchronic repeated-dose toxicity study
Abbreviations: GC-MS, gas chromatography-mass spectrometry; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin; TBA, total bile acid; TG, triglyceride; TC, total cholesterol; GLU, glucose; BUN, blood urea nitrogen; CRE, creatinine; CK, creatine kinase; TP, total protein; ALB, albumin; PCA, principal component analysis; PLS-DA, partial least squares-discriminant analysis; H&E, Hematoxylin and eosin ∗ Corresponding author. Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, No.88 Yuquan Road, Nankai District, Tianjin, 300193, China. E-mail addresses: [email protected], [email protected] (K. Zhou). 1 They are co-first authors. https://doi.org/10.1016/j.yrtph.2019.01.010 Received 18 February 2017; Received in revised form 19 October 2018; Accepted 2 January 2019 Available online 08 January 2019 0273-2300/ © 2019 Published by Elsevier Inc.
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automatic blood biochemical analyzer (HITACHI 7020; Hitachi, Tokyo, Japan) as follows: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), total bile acid (TBA), triglyceride (TG), total cholesterol (TC), glucose (GLU), blood urea nitrogen (BUN), creatinine (CRE), creatine kinase (CK), total protein (TP), and albumin (ALB).
was used to evaluate the safety of long-term treatment with isopsoralen in our study. Additionally, the toxic mechanism of isopsoralen cannot be clarified in a conventional drug preclinical safety study. Thus, a method is needed to solve this problem. Metabolomics is the study of the types, quantity, and metabolic changes of biological endogenous metabolites (Nicholson et al., 1999). It can also provide surprisingly detailed insights into the changes in metabolic processes in whole organisms (Nicholson and Wilson, 2003). Just like genomics, transcriptomics, and proteomics, metabolomics has been gradually proven to be a powerful tool in determining the overall physiologic status of an individual metabolic profile in response to living systems (Wang et al., 2012b). Furthermore, metabolomics is widely used in disease diagnosis and prevention, nutrition, pharmacology, toxicology, botany, and other fields (Zhang et al., 2010; Powers, 2009). In this study, we observed the toxic effects of isopsoralen administration for 3 months in Wistar rats. To explore the possible mechanism of isopsoralen toxicity, and to seek early markers for predicting further toxicity, gas chromatography-mass spectrometry (GC-MS) was applied to detect metabolites in plasma samples.
2.5. Histopathological examination The target organs, including the brain (cerebrum and epencephalon), hypophysis, heart, liver, kidney, spleen, lungs (containing trachea), adrenal glands, thymus, stomach (connected to duodenum), pancreas, jejunum, ileum, colon, testis and epididymis, ovaries, and uterus (connected to vagina), were placed into a fixation medium of 10% formalin solution, followed by dehydration, embedding in paraffin, and sectioning at 5-μm thickness. The organs were examined macroscopically after staining with hematoxylin and eosin (H&E). 2.6. Plasma sample pretreatment
Isopsoralen was purchased from Tianjin Crescent Lake Biotech Co. Ltd. (Tianjin, China). Gum tragacanth powder (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) was used as a fluxing agent when preparing the isopsoralen solution.
A 200-μL plasma sample was added to 600 μL of acetonitrile and mixed. After 5 min of centrifugation at approximately 12,000 rpm (4 °C), the 500-μL supernatant was mixed with 30 mL of pyridine methoxyl amine (40 mg/mL) and incubated at 30 °C for 90 min for derivatization. Next, 100 μL of silicon alkylation reagents was added and the mixture was incubated at 40 °C for 30 min for derivatization. After another centrifugation for 5 min at approximately 12,000 rpm (4 °C), the supernatant was injected into the GC-MS system. The injection volume was 1 μL.
2.2. Experimental animals
2.7. GC-MS analysis
Female and male Wistar rats, weighing 130–150 g, were purchased from the pathogen-free facility at Beijing HFK Bioscience Technology Co. Ltd. (Beijing, China). The rats were housed in the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, and the experimental environment was maintained at 18–22 °C and a humidity of 55%–65%. The rats ate a standard diet and drank water ad libitum. All rats were allowed to adapt to the new environment for 7 days before drug administration. The protocols of the animal experiments were approved by the Laboratory Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (permit number: TCM-LAEC 2015012).
The MS conditions were as follows: ionization mode, EI; electron energy, 70eV; interface temperature, 250 °C; ion source temperature, 230 °C; transmission line temperature, 280 °C; quadrupole temperature, 150 °C; mass scan range, 60–600. The chromatographic column was HP5ms (30 m × 250 μm × 0.25 μm) and the carrier gas was He. The programmed temperature profile was 60 °C–325 °C at a rate of 10 °C/min, followed by maintenance for 10 min. The total analysis time was 37.5 min.
2. Materials and methods 2.1. Drug
2.8. GC-MS data processing The original data was imported into the AMDIS software (Agilent Technologies), and the data containing the deconvolution, integral, and metabolite identification were analyzed using the Fiehn 2013 Metabolomics RTL database. C8-28 fatty acid methyl ester was used to calibrate the retention time.
2.3. Subchronic toxicity treatment A total of 96 rats were randomly divided into four groups: control group, high-dose group (14 mg/kg), medium-dose group (7.0 mg/kg), and low-dose group (3.5 mg/kg), with equal numbers of male and female rats in each group. The rats in the three isopsoralen groups were intragastrically administered with isopsoralen once every day for 3 months, and the rats in the control group were treated with gum tragacanth solution. The general behavior of the rats was observed and recorded daily, and the weight, food consumption, and water consumption of the rats were detected weekly. Finally, the rats were anesthetized and then euthanized. Blood was collected to examine serum biochemical parameters, and the brain, heart, liver, spleen, kidney, adrenal glands, thymus, testis, epididymis, ovaries, and uterus were tested for visceral coefficients.
2.9. Metabolomic analysis Fold change analysis, principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and pathway analysis were performed using metaboanlayst 3.0 (http://www.metaboanalyst. ca/). The peak data were subjected to log transformation and Pareto scaling (mean-centered and divided by square root of standard deviation of each variable). The metabolites with significant changes induced by isopsoralen were evaluated as a VIP (variable importance in projection) of more than 1 and a P-value of less than 0.05 (t-test) between the control and isopsoralen groups. Finally, the biomarkers were interpreted using relevant biochemical databases, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG: http://www.kegg.com/).
2.4. Measurement of serum biochemical parameters Blood samples were collected, and placed into non-heparinized tubes for separation of serum. After 1 h, the solidified blood samples were centrifuged at 3500 rpm (10 min, 4 °C) and the supernatants were collected. Several serum biochemical parameters were detected by an
2.10. Statistical analysis The data were formulated as the mean ± SD for the tables and 2
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Fig. 1. Effects of repeated administration of isopsoralen on body weight in rats treated for 3 months. A) Female rats. B) Male rats. Data represent mean ± SEM.*P < 0.05, **P < 0.01, significant difference from control rats.
mean ± SEM for the figures. The significance of differences between the groups was evaluated by one-way analysis of variance (ANOVA) and Dunnett's t-test. Differences were considered statistically significant at P < 0.05.
Table 4 shows the serum biochemical parameters in male rats. Compared with the control group, TP activity was increased in all three groups treated with isopsoralen (P < 0.05 or P < 0.001). The levels of AST and BUN increased significantly in the 3.5 mg/kg and 14 mg/kg groups (P < 0.05, P < 0.01 or P < 0.001). In the 14 mg/kg group, the levels of TG, GLU, and CRE decreased significantly (P < 0.05), while the levels of TC, TBA, TG, and ALP in the 7 mg/kg dose group also decreased significantly (P < 0.05).
3. Results 3.1. Body weight, food intake and water intake After treatment with isopsoralen for 3 months, no rats died in the three dose groups. The color, appearance, and behavior of the rats were normal during the experimental period. The body weight of the rats in each group increased by varying degrees, and from week 7, the weights of the female rats treated with 14 mg/kg isopsoralen were lower than those in the control group (Fig. 1). Compared with the control group, the food and water consumption in the 14 mg/kg group decreased, but no significant difference was noted. The food and water consumption of mice in the isopsoralen groups showed no systematic variations (Fig. 2).
3.4. Histopathology In the present study, no abnormal gross pathology was observed during dissection. Some individual pathological changes were observed in the isopsoralen groups by microscopic examination. Compared with the control group, there were multiple points (more than 15) of necrosis or focal necrosis of hepatocytes (Fig. 3A) in the liver of the isopsoralen-treated rats, especially in the medium-dose and low-dose groups. In the pancreatic tissues, infiltration of inflammatory cells, mainly lymphocytes, was observed in the interstitium and arterioles of the pancreas (Fig. 3B) only in female rats in the 3.5 mg/kg (1/12) and 7.0 mg/kg (1/12) groups, while focal or lamellar vacuolar degeneration of the acinar (Fig. 3C) was observed in male rats in the 3.5 mg/kg (3/ 12) and 7.0 mg/kg (4/12) groups. In the male rat reproductive system, there were no appreciable changes in the control group, while the spermatogonium of the seminiferous tubule was degenerated, missing, or necrotic (Fig. 3D) in male rats in the 3.5 mg/kg (1/12) and 7.0 mg/kg (4/12) groups.
3.2. Visceral coefficients Table 1 shows the effects of isopsoralen on the visceral coefficients in female rats. Compared with the control group, the heart coefficient decreased significantly (P < 0.01) and the uterus coefficient increased significantly (P < 0.05) in the high-dose group (14 mg/kg). The ovary coefficient in the middle-dose group (7.0 mg/kg) increased significantly (P < 0.01). The thymus coefficient increased significantly (P < 0.05) and the adrenal gland coefficient also increased significantly (P < 0.05) in the low-dose group (3.5 mg/kg). Table 2 shows the effects of isopsoralen on the visceral coefficients in male rats. Compared with the control group, the testis coefficient in all groups treated with isopsoralen increased significantly (P < 0.05), and the heart coefficient in the 7.0 mg/kg group and the adrenal gland coefficient in the 14 mg/kg group also increased significantly (P < 0.05). No significant changes were observed in other visceral coefficients.
3.5. Metabolomic analyses in rat plasma Based on the m/z values and fragmentation patterns, 77 metabolites were identified in the GC-MS spectra of 24 plasma samples collected from the control and high-dose isopsoralen groups. The commonly used unsupervised multivariate statistical analysis (MVA) method PCA was used to examine the metabolomics data. The three-dimensional (3D) PCA score plots (Fig. 4A and B) indicated the dataset was of an acceptable quality and homogeneity without any outliers. Supervised PLS-DA was subsequently used to identify the differential features that were associated with the observed toxicities of isopsoralen on rats. An obvious separation between the female rats in the control and treated groups was observed in the PLS-DA score plot (Fig. 4C) with the class discrimination statistical parameters R2 of 0.9996 and Q2 of 0.6703. Variables contributing to the class clustering and discrimination were extracted via two criterions, including a value of the variable importance in the project (VIP) of more than 1 and p values from the ttest of less than 0.05. As a result, 19 metabolites were selected to
3.3. Blood biochemical parameters Several clinical parameters in serum were measured to monitor the toxic effects of isopsoralen. Table 3 shows the serum biochemical parameters in female rats. Serum GLU and CRE in all dose groups decreased significantly compared with the control group (P < 0.05 or P < 0.001). Other serum biochemical parameters were not significantly changed, except for a significant decrease in AST in the 3.5 mg/kg group (P < 0.01), and significant increases in ALP (P < 0.001) and BUN (P < 0.05). 3
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Fig. 2. Effects of repeated administration of isopsoralen on food and water intake in rats treated for 3 months. A) Food intake of female rats. B) Food intake of male rats.C) Water intake of female rats. D) Water intake of male rats. Data represent mean ± SEM.
Table 1 Effects of repeated administration of isopsoralen on visceral coefficients in female rats treated for 3 months (mean ± SD, n = 12). viscera coefficient
Control
brain heart thymus liver kidney adrenal glands spleen ovaries uterus
0.6074 0.3552 0.1257 2.5901 0.5862 0.0248 0.1805 0.0534 0.1821
± ± ± ± ± ± ± ± ±
0.0482 0.0308 0.0194 0.1442 0.0423 0.0028 0.0200 0.0087 0.0599
3.5 mg/kg
7.0 mg/kg
14 mg/kg
0.6149 0.3737 0.1487 2.6202 0.6075 0.0227 0.1728 0.0509 0.1802
0.6190 0.3450 0.1325 2.4920 0.6072 0.0229 0.1896 0.0617 0.1877
0.6317 0.3209 0.1222 2.4963 0.6173 0.0237 0.1782 0.0559 0.2598
± 0.0439 ± 0.0339 ± 0.0344* ± 0.2327 ± 0.0486 ± 0.0030* ± 0.0582 ± 0.0070 ± 0.0477
± 0.0396 ± 0.0284 ± 0.0276 ± 0.1087 ± 0.0403 ± 0.0029 ± 0.0395 ± 0.0068** ± 0.0647
± 0.0451 ± 0.0276** ± 0.0162 ± 0.0982 ± 0.0437 ± 0.0023 ± 0.0241 ± 0.0065 ± 0.1084*
*P < 0.05, **P < 0.01, significant difference from the control group. Table 2 Effects of repeated administration of isopsoralen on visceral coefficients in male rats treated for 3 months (mean ± SD, n = 12). viscera coefficient
Control
brain heart thymus liver kidney adrenal glands spleen testis epididymis
0.3969 0.3131 0.0881 2.2803 0.5626 0.0113 0.1706 0.7304 0.3050
± ± ± ± ± ± ± ± ±
0.0308 0.0277 0.0233 0.1526 0.0589 0.0021 0.0660 0.0550 0.0198
3.5 mg/kg
7.0 mg/kg
14 mg/kg
0.4122 0.3304 0.0999 2.4132 0.5880 0.0129 0.1739 0.8112 0.3094
0.4091 0.3454 0.1067 2.3367 0.5766 0.0130 0.1660 0.8028 0.2890
0.4071 0.3325 0.1024 2.3526 0.5698 0.0132 0.1558 0.8048 0.3053
± 0.0265 ± 0.0217 ± 0.0240 ± 0.3714 ± 0.0557 ± 0.0018 ± 0.0686 ± 0.0728* ± 0.0370
*P < 0.05, significant difference from the control group.
4
± 0.0361 ± 0.0295** ± 0.0197 ± 0.2032 ± 0.0400 ± 0.0021 ± 0.0334 ± 0.0695* ± 0.0397
± 0.0293 ± 0.0276 ± 0.0272 ± 0.1159 ± 0.0531 ± 0.0017* ± 0.0168 ± 0.0830* ± 0.0247
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Table 3 Effects of repeated administration of isopsoralen on blood biochemical parameters in female rats treated for 3 months (mean ± SD, n = 12). parameter
Control
3.5 mg/kg
7.0 mg/kg
14 mg/kg
ALT AST ALP TBIL TBA TG TC GLU BUN CRE CK TP ALB
26.18 ± 5.02 156.45 ± 28.73 49.36 ± 13.46 0.14 ± 0.03 70.12 ± 23.26 0.38 ± 0.11 1.91 ± 0.49 10.82 ± 2.25 5.18 ± 0.99 92.34 ± 8.76 1280.46 ± 1145.69 64.75 ± 2.83 28.37 ± 2.61
24.27 ± 5.78 131.18 ± 19.45* 81.55 ± 29.85*** 0.11 ± 0.04 82.83 ± 23.38 0.37 ± 0.15 2.03 ± 0.27 8.64 ± 2.19* 6.17 ± 1.25* 77.80 ± 4.95*** 771.73 ± 614.21 66.46 ± 3.43 27.59 ± 2.43
29.83 ± 14.80 148.67 ± 26.58 58.25 ± 20.04 0.18 ± 0.07 83.37 ± 40.64 0.43 ± 0.16 1.96 ± 0.30 7.18 ± 2.09*** 5.33 ± 1.02 61.61 ± 6.04*** 978.92 ± 419.41 65.95 ± 2.91 28.36 ± 2.19
23.80 ± 5.71 135.40 ± 21.20 50.00 ± 12.18 0.16 ± 0.06 80.52 ± 36.04 0.38 ± 0.15 2.19 ± 0.37 7.39 ± 1.64*** 5.04 ± 0.76 64.69 ± 4.72*** 1176.80 ± 800.81 66.80 ± 4.09 27.79 ± 1.51
*P < 0.05, **P < 0.01, ***P < 0.001, significant difference from the control group. Table 4 Effects of repeated administration of isopsoralen on blood biochemical parameters in male rats treated for 3 months (mean ± SD, n = 12). parameter
Control
3.5 mg/kg
7.0 mg/kg
14 mg/kg
ALT AST ALP TBIL TBA TG TC GLU BUN CRE CK TP ALB
42.67 ± 14.20 205.40 ± 52.41 107.90 ± 26.86 0.20 ± 0.08 126.86 ± 33.72 0.44 ± 0.17 1.60 ± 0.27 7.22 ± 1.99 5.92 ± 0.80 85.42 ± 10.88 2307.20 ± 1472.66 59.48 ± 1.63 23.56 ± 0.99
35.90 ± 13.00 146.22 ± 25.21*** 93.50 ± 16.92 0.15 ± 0.06* 119.51 ± 45.57 0.42 ± 0.11 1.69 ± 0.28 6.90 ± 3.37 5.10 ± 0.76* 79.08 ± 8.47 2023.20 ± 1333.42 62.77 ± 4.83* 23.82 ± 2.09
33.45 ± 11.92 203.00 ± 44.25 79.09 ± 14.27** 0.15 ± 0.05 87.82 ± 38.27* 0.30 ± 0.10* 1.31 ± 0.24* 6.79 ± 1.79 5.66 ± 0.69 81.89 ± 9.24 2403.27 ± 999.59 61.30 ± 1.69* 23.43 ± 1.34
35.64 ± 12.86 143.82 ± 20.50** 92.00 ± 23.14 0.16 ± 0.06 96.00 ± 50.89 0.29 ± 0.08* 1.46 ± 0.33 5.37 ± 1.10* 5.15 ± 0.61* 78.14 ± 6.46* 1335.91 ± 545.37* 64.13 ± 2.22*** 24.22 ± 0.80
*P < 0.05, **P < 0.01, ***P < 0.001, significant difference from the control group.
Fig. 3. Representative histopathological photographs of organ lesions in Wistar rats. A) Liver, with hepatocyte focal necrosis. B) Pancreas, with inflammatory cell infiltration.C) Pancreas, with vacuolar degeneration of the acinar. D) Testicle, with degeneration and necrosis of the spermatogonium. H&E staining, 200×.
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Fig. 4. PCA and PLS-DA score plots of plasma samples in the control and isopsoralen groups according to sex. A) PCA score plot of female rats. B) PCA score plot of male rats. C) PLS-DA score plot of female rats. D) PLS-DA score plot of male rats.
commonly affected by isopsoralenare intervention in both male and female rats, while the other metabolites were completely different. With an impact factor > 0.1 and raw p value < 0.1, the pathway topology and integrating enrichment analyses (Fig. 5B and C) showed that the metabolic changes induced by isopsoralen also had significant gender differences in rats. Aside from two common pathways (glycine, serine, and threonine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis), isopsoralen disturbed another six metabolism pathways (aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, pyruvate metabolism, alanine, aspartate and glutamate metabolism, D-glutamine and D-glutamate metabolism and linoleic acid metabolism) in female rats and four other pathways (glyoxylate and dicarboxylate metabolism, citrate cycle, glycerolipid metabolism, cysteine and methionine metabolism) in male rats.
indicate the impact of isopsoralen on the metabolism of female rats. Compared with the control group, the levels of pyruvic acid, L-lactic acid, 2-ketobutyric acid, oxalic acid, D-threitol, L-glutamic acid, fructose, L-lysine, tyrosine, and serotonin were up-regulated, while the levels of L-leucine, phosphoric acid, L-serine, palmitic acid, heptadecanoic acid, stearic acid, linoleic acid, alpha tocopherol, and cholesterol were down-regulated in the female administration group (Table 5). A PLS-DA model was also established to classify the samples from the control and treated male rats. The score plot (Fig. 4D) displayed a clustering trend for different groups with an R2 of 0.9980 and Q2 of 0.6505. Through the abovementioned criterions, 28 metabolites were extracted as the differential features for revealing the metabolic changes induced by isopsoralen on male rats. Compared with the control group, the content of indole 3-propionic acid was decreased in the male administration group, while the other 27 metabolites were increased (Table 6). Venn analysis (Fig. 5A) revealed that only three metabolites were 6
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2012a). Isopsoralen is a major active and quality-control component of Fructus Psoraleae in the Chinese pharmacopoeia and its pharmacological activities have been investigated. Interestingly, a study revealed that some compounds of Fructus Psoraleae were metabolized to isopsoralen by intestinal microflora through deglucosylation (Wang et al., 2014), indicating that the concentration of isopsoralen may be much higher in vivo. Therefore, aside from analyzing its historical and current applications, toxicity studies are urgently needed to assess the safety of the wide array of isopsoralen doses in use. Changes in body weight is the most basic indicator of toxicity toward organs and systems (Thinkratok et al., 2014; Santos et al., 2009). As shown in Fig. 1, from week 7 to the end of administration, the body weight gains in the 14 mg/kg dose group were significantly decreased in female rats compared with male rats. Furthermore, an increase in a visceral coefficient is an indication of cell swelling or inflammation, while a reduction in the same parameter can be attributed to cellular constriction (AOT Ashafa et al., 2009). In the present study, the heart coefficient of female rats in the isopsoralen groups was significantly lower than that in the control group, and the ovary coefficient was significantly higher. Additionally, the testis, heart, and adrenal gland coefficients of male rats were apparently increased compared with the control group. These findings imply that isopsoralen may have effects on the reproductive system in both female and male rats. Alterations in AST, ALP, and histopathologic features of the liver occurred in rats receiving treatment with isopsoralen. Serum levels of ALT, AST, and ALP are commonly used as markers of liver damage (Ozer et al., 2008). In our study, serum AST was significantly decreased and serum ALP was increased in female rats in the 3.5 mg/kg isopsoralen group, while serum AST and ALP were lower in male rats in all groups compared with the control rats. Additionally, the liver is the site of cholesterol disposal or degradation, is a major site of synthesis (Cofan Pujol, 2014), and it was observed that serum TC and TG were lower in male rats treated with isopsoralen compared with the control group. Decreased CRE in female rats was correlated with toxic changes in the kidney. Decreased GLU in female rats may be correlated with severe malnutrition, but we cannot make a conclusion with regards as to why the GLU decreased in this study. The BUN was increased in female rats treated with 14 mg/kg isopsoralen. In male rats, BUN was significantly decreased in the 3.5 and 14 mg/kg isopsoralen groups. Our results demonstrate that isopsoralen may have toxic effects compared with the control group. It is worth mentioning that such changes were not consistent over time or dose-related, and only appeared at the low dose. This phenomenon has been previously observed in some studies (Fagin, 2012), and researchers suggest that certain chemicals can have toxic effects at very low doses. To better observe the toxic effects of isopsoralen in rats, a metabolomic study of the biochemical profiles was performed by integrating GC-MS analyses. In our study, we found that the toxic effects of isopsoralen were sexdependent, with the hepatotoxicity and nephrotoxicity in female rats appearing to be more significant than those in male rats. Therefore, we performed PCA and PLS-DA to analyze and compare the metabolic profiles by sex. A total of 19 and 28 metabolites were significantly changed in the plasma samples from female rats and male rats, respectively. A metabolomic analysis is suitable for direct assessment of isopsoralen toxicity, and the biological relationships between the different metabolites and toxic effects induced by isopsoralen in both male and female rats are discussed. The disturbance of metabolic pathways primarily occurred in those involved in amino acid metabolism, regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Regarding phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine is converted to tyrosine in the liver by phenylalanine hydroxylase (Waters et al., 2000). An accumulation of phenylalanine in the blood leads to phenylketonuria (Al Hafid and Christodoulou, 2015). In our research, based on the findings of increased phenylalanine in male rats and decreased tyrosine in female rats (Fig. 6A), we propose that isopsoralen
Table 5 Significant alterations of plasma metabolites in female rats treated with isopsoralen (14 mg/kg). Metabolites
R.T.(min)
t- test
VIP
Trend
pyruvic acid L- lactic acid 2-Ketobutyric acid oxalic acid L-leucine phosphoric acid L-serine D-threitol L-glutamic acid fructose L-lysine tyrosine palmitic acid heptadecanoic acid stearic acid Linoleic acid serotonin alpha tocopher cholesterol
6.158 6.327 6.884 7.347 9.342 9.455 10.574 12.532 13.771 16.705 17.011 17.211 18.062 18.963 19.513 19.582 21.834 26.582 26.719
0.0013073 0.032428 0.048028 0.035542 0.044273 0.0035397 0.042941 0.0025924 0.023179 0.020118 0.011119 0.003015 0.026962 0.0029989 0.010864 0.029956 0.040543 0.034899 0.0007268
2.0809 1.3207 1.6059 1.3698 1.1499 1.9766 1.2278 1.6037 1.4477 1.511 1.7113 2.2409 1.1695 1.5662 1.3406 1.4944 1.7499 1.0666 1.4628
down down down down up up up down down down down down up up up up down up up
Table 6 Significant alterations of plasma metabolites in male rats treated with isopsoralen (14 mg/kg). Metabolites
R.T.(min)
t- test
VIP
Trend
indole 3-propionic acid pyruvic acid glycolic acid acetohydroxamic acid 2-hydroxybutyric acid oxalic acid N-methylalanine 2-ketoisocaproic acid ethanolamine caprylic acid glycerol phosphoric acid glycine glyceric acid fumaric acid tartronic acid L-methionine Beta-alanine D-malic acid phenylalanine L-pyroglutamic acid hypotaurine L-fucose O-phosphocolamine citric acid gluconic acid mucic acid myo-inositol
2.6142 6.158 6.521 7.09 7.284 7.347 7.922 8.523 9.261 9.28 9.405 9.455 9.824 10.187 10.293 10.38 11.137 11.4 12.22 12.851 12.601 13.539 15.128 15.654 16.048 17.655 18.143 18.806
0.025856 0.0013097 0.00085709 0.036681 0.0031334 0.02375 0.0022555 0.020824 0.0049312 0.0024793 0.0066685 1.88E-05 0.0027673 0.00010793 0.00061052 1.53E-05 0.0020704 0.0060127 0.00075731 0.011092 0.014067 0.006016 0.0062063 0.044344 0.00028363 0.0038772 0.0059397 0.0068841
1.173 1.6191 1.5738 1.2263 1.3283 1.2431 1.1775 1.1385 1.527 2.021 1.5146 1.3138 1.428 1.9922 1.7443 1.7673 1.3465 1.3106 1.7768 1.1014 1.375 1.0495 1.2324 1.1952 1.5757 1.6964 1.2756 1.225
down up up up up up up up up up up up up up up up up up up up up up up up up up up up
4. Discussion Traditional Chinese Medicine is widely used to cure and prevent various diseases, but there remain problems with its clinical use. Recently, the potential for adverse effects of Traditional Chinese Medicine in clinical usage has become an important issue (Lai et al., 2013). Fructus Psoraleae has been widely used for its therapeutic effects on osteoporosis, osteomalacia, bone fracture, vitiligo, and psoriasis. Nowadays, many clinical reports on adverse reactions of Fructus Psoraleae have caused social concern (Cheung et al., 2009). Toxicology studies on animals suggest that Fructus Psoraleae mainly exhibits toxicity toward the liver (Chitturi and Farrell, 2008; Wang et al.,
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Fig. 5. Pathway analyses for differential metabolites in plasma. A) Venn analysis of the metabolic biomarkers induced by isopsoralen on female and male rats; B) Bubble plot of the metabolism pathways disturbed by isopsoralen on female rats; C) Bubble plot of the metabolism pathways disturbed by isopsoralen on male rats. Fig. 6. Pathway analyses for plasma metabolites. A) phenylalanine, tyrosine, and tryptophan biosynthesis; B) glycine, serine and threonine metabolism. The metabolites with blue arrows were changed in male rats and the metabolites with red arrows were changed in female rats. (↑) indicates that the compound was upregulated in the model group and (↓) indicates that the compound was downregulated in the model group.
in mice, and Wang et al. (2016) studied urine metabolomics for the blood-replenishing mechanism of Angelica sinensis in a blood-deficient mouse model. These analyses may have ignored differential efficacies or toxicities based on sex. In fact, sex differences in human and animal toxicities have raised much concern (Gochfeld, 2007). Sex-dependent metabolism often leads to differences in pharmacological or toxicological responses to xenobiotics (Mugford and Kedderis, 1998). Sexdependent metabolic variations in Wistar rats were confirmed by Stanley et al. (2005) using 1H nuclear magnetic resonance spectroscopy of urine coupled with chemometric methods. In our study, we found that isopsoralen showed sex differences in its toxicities and plasma metabolomics. Compared with the control group, female rats were found to be more sensitive to isopsoralen than male rats, and the body weight gains of female rats in the 3.5 mg/kg dose group were
may affect the metabolic function of the liver. For glycine, serine, and threonine metabolism, glycine and serine have multiple physiological functions in animals and humans. Glycine is a major component of collagen and elastin, as the most abundant proteins in the body (Wu, 2009). Serine hydroxymethyltransferase catalyzes the formation of glycine from serine (Wang et al., 2013). In our research, markedly higher levels of glyceric acid and glycine were detected in male rats, as well as a markedly higher level of L-serine in female rats after isopsoralen treatment (Fig. 6B). Thus, isopsoralen may cause amino acid imbalances and toxicity. In many metabolism studies, the experiments were only performed on male animals. For example, Song et al. (2015) used GC/MS-based metabolomic profiling to study the therapeutic effects of a Huangbai–Zhimu herb-pair extract on streptozotocin-induced type 2 diabetes 8
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suppressed, especially during the last few weeks. Additionally, with respect to blood biochemical parameters, female rats appeared to be more sensitive to hepatotoxicity and nephrotoxicity than male rats after treatment with isopsoralen. Moreover, based on the analysis of metabolites, there were 19 metabolic differences in female rats and 28 metabolic differences in male rats after isopsoralen administration. As shown in Fig. 5, the primary affected pathways in female rats and male rats all included those involved in amino acid metabolism, but the potential biomarkers involved in the pathways were different (Fig. 6). Furthermore, fatty acid metabolism, such as linoleic acid metabolism and methane metabolism, were the main affected pathways in female rats, while lipid metabolism, such as glycerolipid metabolism, and energy metabolism, such as glyoxylate and dicarboxylate metabolism, were the main affected pathways in male rats. Thus, future studies should use balanced sexes and analyze data by sex and other interactions rather than simply ignoring the effect of sex. Further investigations are still needed to uncover the mechanisms of the sex differences in isopsoralen-induced toxicity.
683–685. Chitturi, S., Farrell, G.C., 2008. Hepatotoxic slimming aids and other herbal hepatotoxins. J. Gastroenterol. Hepatol. 23 (3), 366–373. Citarasu, T., et al., 2003. Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae. Aquacult. Int. : journal of the European Aquaculture Society 11, 583–595. Cofan Pujol, M., 2014. Mecanismos básicos. Absorción y excreción de colesterol y otros esteroles. Clín. Invest. Arterioscler. 26, 41–47. Fagin, D., 2012. Toxicology: the learning curve. Nature 490, 462–465. Feng, C.-Y., et al., 2014. Mitochondrial proteomic analysis of ecdysterone protection against oxidative damage in human lens epithelial cells. Int. J. Ophthalmol. 7, 38–43. Gochfeld, M., 2007. Framework for gender differences in human and animal toxicology. Environ. Res. 104, 4–21. Haraguchi, H., et al., 2002. Antioxidative components of Psoralea corylifolia (leguminosae) phytotherapy research volume 16, issue 6. Phytother Res. 16, 539–544. Khatune, N.A., et al., 2004. Antibacterial compounds from the seeds of Psoralea corylifolia. Fitoterapia 75, 228–230. Lai, J.-N., et al., 2013. Observational studies on evaluating the safety and adverse effects of traditional Chinese medicine. Evid. Based Complement Altern. Med. 2013, 1–9. Lim, S.-H., et al., 2009. Ethanol extract of Psoralea corylifolia L. and its main constituent, bakuchiol, reduce bone loss in ovariectomised Sprague-Dawley rats. Br. J. Nutr. 101, 1031–1039. Mar, W., et al., 2001. Cytotoxic constituents of Psoralea corylifolia. Arch Pharm. Res. (Seoul) 24, 211–213. Mugford, C.A., Kedderis, G.L., 1998. Sex-dependent metabolism of xenobiotics. Drug Metab. Rev. 30, 441–498. Nam, S.W., et al., 2005. A case of acute cholestatic hepatitis associated with the seeds of Psoralea corylifolia (Boh-Gol-Zhee). Clin. Toxicol. 43, 589–591. Nicholson, J.K., et al., 1999. 'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica; the fate of foreign compounds in biological systems 29, 1181–1189. Nicholson, J.K., Wilson, I.D., 2003. Understanding 'global' systems biology: metabonomics and the continuum of metabolism. Nat. Rev. Drug Discov. 2, 668–676. Ozer, J., et al., 2008. The current state of serum biomarkers of hepatotoxicity. Toxicology 245, 194–205. Powers, R., 2009. NMR metabolomics and drug discovery. Magn. Reson. Chem. 47, S2–S11. Santos, S.R., et al., 2009. Toxicological and phytochemical studies of Aspidosperma subincanum Mart. stem bark (Guatambu). Pharmazie 64, 836–839. Song, L., et al., 2015. Application of GC/MS-based metabonomic profiling in studying the therapeutic effects of Huangbai–Zhimu herb-pair (HZ) extract on streptozotocin-induced type 2 diabetes in mice. J. Chromatogr. B 997, 96–104. Stanley, E.G., et al., 2005. Sexual dimorphism in urinary metabolite profiles of Han Wistar rats revealed by nuclear-magnetic-resonance-based metabonomics. Anal. Biochem. 343, 195–202. Tang, S.Y., et al., 2004. Characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional Chinese medicines. Free Radic. Biol. Med. 36, 1575–1587. Thinkratok, A., et al., 2014. Safety assessment of hydroethanolic rambutan rind extract: acute and sub-chronic toxicity studies. Indian J. Exp. Biol. 52, 989–995. Tsai, M.-H., et al., 2007. Psoralea corylifolia extract ameliorates experimental osteoporosis in ovariectomized rats. Am. J. Chin. Med. 35, 669–680. Wang, J., et al., 2012a. Evaluation of hepatotoxicity and cholestasis in rats treated with EtOH extract of Fructus Psoraleae. J. Ethnopharmacol. 144, 73–81. Wang, T., et al., 2016. Urine metabonomic study for blood-replenishing mechanism of Angelica sinensis in a blood-deficient mouse model. Chin. J. Nat. Med. 14, 210–219. Wang, W., et al., 2013. Glycine metabolism in animals and humans: implications for nutrition and health. Amino Acids 45, 463–477. Wang, X., et al., 2012b. Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease. Mol. Cell. Proteomics : MCP. 11, 370–380. Wang, Y.-F., et al., 2014. A UPLC–MS/MS method for in vivo and in vitro pharmacokinetic studies of psoralenoside, isopsoralenoside, psoralen and isopsoralen from Psoralea corylifolia extract. J. Ethnopharmacol. 151, 609–617. Wang, Y., et al., 2011. Screening antitumor compounds psoralen and isopsoralen fromPsoralea corylifoliaL. Seeds. Evid. Based Complement Altern. Med. 2011, 1–7. Waters, P.J., et al., 2000. Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype. Mol. Genet. Metabol. 69, 101–110. Wu, G., 2009. Amino acids: metabolism, functions, and nutrition. Amino Acids 37, 1–17. Yuan, X., et al., 2016. Psoralen and isopsoralen ameliorate sex hormone deficiency-induced osteoporosis in female and male mice. BioMed Res. Int. 2016, 6869452. Zhang, A., et al., 2010. Metabolomics: towards understanding traditional Chinese medicine. Planta Med. 76, 2026–2035. Zhang, C.Z., et al., 2005. In vitro estrogenic activities of Chinese medicinal plants traditionally used for the management of menopausal symptoms. J. Ethnopharmacol. 98, 295–300.
5. Conclusions This study revealed the impact of isopsoralen by conducting subchronic toxicity experiments and employing GC/MS-based metabolomic profiling combined with multivariate statistical analysis. Visceral coefficients and serum biochemical parameters were significantly changed after isopsoralen administration. Female rats were more sensitive to isopsoralen-induced toxicity than male rats since the weights of the female rats significantly decreased and the infiltration of inflammatory cells was observed in the interstitium and arterioles of the pancreas only in female rats. The liver, pancreas, and reproductive system may be the target organs affected by isopsoralen based on the histopathological findings. The metabolic analysis of plasma samples revealed that isopsoralen disturbed the metabolism of amino acids. We also found that isopsoralen exhibited sex differences in its toxicities and plasma metabolomics. The mechanism of isopsoralen toxicity and its observed sex differences as identified by metabonomics in this study requires further research to confirm the pathways involved. Conflicts of interest The authors declare that there are no conflicts of interest in regards to this manuscript. Acknowledgments The work was supported by the National Natural Science Foundation of China (No.81202991, 816738261, 81703790). We thank Wei Nie and Hong Shi for their help in this work. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.yrtph.2019.01.010. References AI Hafid, N., Christodoulou, J., 2015. Phenylketonuria: a review of current and future treatments. Transl. Pediatr. 4, 304–317. AOT Ashafa, M.Y., Grierson, D.S., Afolayan, A.J., 2009. Toxicological evaluation of the aqueous extract of Felicia muricata Thunb. leaves in Wistar rats. Afr. J. Biotechnol. 8 (6), 949–954. Cheung, W.I., et al., 2009. Liver injury associated with the use of Fructus Psoraleae (Bolgol-zhee or Bu-gu-zhi) and its related proprietary medicine. Clin. Toxicol. 47,
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Isopsoralen induces different subchronic toxicities and metabolomic outcomes between male and female Wistar rats
T
Yue Zhanga,b,c,1, Xiao-mei Yuana,1, Yue-fei Wanga,b, Miao-miao Jianga,b, Ya-nan Bia, Ying Liud, Wei-ling Pua, Lei Songa,c, Ju-yang Huanga,c, Li-kang Suna,b, Zhi-xing Zhoue, Kun Zhoua,b,c,∗ a
Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin, 300193, China c Ministry of Education Key Laboratory of Traditional Chinese Medical Formulae, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China d College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, 010018, China e Tianjin Institute of Pharmaceutical Research, Tianjin, 300193, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Wistar rats Fructus psoraleae Isopsoralen Subchronic toxicity Metabolomics
Isopsoralen is a major active and quality-control component of Fructus Psoraleae, but lacks a full safety evaluation. We evaluated the oral toxicity of isopsoralen in Wistar rats treated for 3 months at doses of 0, 3.5, 7.0, and 14 mg/kg. Additionally, the plasma metabolomics of isopsoralen in male and female rats treated for 3 months at doses of 0 and 14 mg/kg were investigated by gas chromatography-mass spectrometry. Many abnormalities were observed in the isopsoralen-treated rats, including suppression of body weight gain, and changes in serum biochemical parameters and visceral coefficients. Histopathological changes in liver, pancreatic, and reproductive system tissues were also observed in the isopsoralen-treated rats. The metabolomic analyses showed alterations in many metabolites (19 in female rats; 28 in male rats) after isopsoralen administration. The significant changes in these metabolites revealed metabolomic alterations in the isopsoralentreated rats, especially in amino acid metabolism regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Furthermore, fatty acid metabolism comprised the main affected pathways in female rats, while lipid metabolism and energy metabolism were the main affected pathways in male rats.
1. Introduction Fructus Psoraleae, the seed of Psoralea corylifolia L., has been used extensively in China for its various biological activities, including antioxidant activity (Haraguchi et al., 2002; Tang et al., 2004), antimicrobial activity (Khatune et al., 2004; Citarasu et al., 2003), osteoblastic activity (Lim et al., 2009; Tsai et al., 2007), and estrogen-like effect (Zhang et al., 2005). Some studies have revealed the hepatotoxicity and cytotoxicity of Fructus Psoraleae in recent years (Nam et al., 2005; Cheung et al., 2009)(Mar et al., 2001). Isopsoralen is one of the two quality-control components of Fructus Psoraleae according to the Chinese Pharmacopoeia (2015 edition). The pharmacological activities of isopsoralen have been widely researched. One study showed
that isopsoralen had a protective effect against oxidative damage in human lens epithelial cells (Feng et al., 2014). Another study suggested that isopsoralen significantly inhibited the proliferation of sensitive and multidrug-resistant cancer cells, and had anticancer activity in vitro (Wang et al., 2011). Isopsoralen also exhibited osteoprotective effects in ovariectomized and orchidectomized mice (Yuan et al., 2016). However, few studies on the toxicity or safety evaluations of isopsoralen have been reported. One exception was a report that ingestion of isopsoralen for 28 days may influence metabolism and excretion in mice (Wang et al., 2012b #46), but there are no reliable predictive and prognostic biomarkers to assess the hepatotoxicity induced by isopsoralen. Clearly, not only the therapeutic effect, but also the safety of a drug is important, and thus a subchronic repeated-dose toxicity study
Abbreviations: GC-MS, gas chromatography-mass spectrometry; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin; TBA, total bile acid; TG, triglyceride; TC, total cholesterol; GLU, glucose; BUN, blood urea nitrogen; CRE, creatinine; CK, creatine kinase; TP, total protein; ALB, albumin; PCA, principal component analysis; PLS-DA, partial least squares-discriminant analysis; H&E, Hematoxylin and eosin ∗ Corresponding author. Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, No.88 Yuquan Road, Nankai District, Tianjin, 300193, China. E-mail addresses: [email protected], [email protected] (K. Zhou). 1 They are co-first authors. https://doi.org/10.1016/j.yrtph.2019.01.010 Received 18 February 2017; Received in revised form 19 October 2018; Accepted 2 January 2019 Available online 08 January 2019 0273-2300/ © 2019 Published by Elsevier Inc.
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automatic blood biochemical analyzer (HITACHI 7020; Hitachi, Tokyo, Japan) as follows: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), total bile acid (TBA), triglyceride (TG), total cholesterol (TC), glucose (GLU), blood urea nitrogen (BUN), creatinine (CRE), creatine kinase (CK), total protein (TP), and albumin (ALB).
was used to evaluate the safety of long-term treatment with isopsoralen in our study. Additionally, the toxic mechanism of isopsoralen cannot be clarified in a conventional drug preclinical safety study. Thus, a method is needed to solve this problem. Metabolomics is the study of the types, quantity, and metabolic changes of biological endogenous metabolites (Nicholson et al., 1999). It can also provide surprisingly detailed insights into the changes in metabolic processes in whole organisms (Nicholson and Wilson, 2003). Just like genomics, transcriptomics, and proteomics, metabolomics has been gradually proven to be a powerful tool in determining the overall physiologic status of an individual metabolic profile in response to living systems (Wang et al., 2012b). Furthermore, metabolomics is widely used in disease diagnosis and prevention, nutrition, pharmacology, toxicology, botany, and other fields (Zhang et al., 2010; Powers, 2009). In this study, we observed the toxic effects of isopsoralen administration for 3 months in Wistar rats. To explore the possible mechanism of isopsoralen toxicity, and to seek early markers for predicting further toxicity, gas chromatography-mass spectrometry (GC-MS) was applied to detect metabolites in plasma samples.
2.5. Histopathological examination The target organs, including the brain (cerebrum and epencephalon), hypophysis, heart, liver, kidney, spleen, lungs (containing trachea), adrenal glands, thymus, stomach (connected to duodenum), pancreas, jejunum, ileum, colon, testis and epididymis, ovaries, and uterus (connected to vagina), were placed into a fixation medium of 10% formalin solution, followed by dehydration, embedding in paraffin, and sectioning at 5-μm thickness. The organs were examined macroscopically after staining with hematoxylin and eosin (H&E). 2.6. Plasma sample pretreatment
Isopsoralen was purchased from Tianjin Crescent Lake Biotech Co. Ltd. (Tianjin, China). Gum tragacanth powder (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) was used as a fluxing agent when preparing the isopsoralen solution.
A 200-μL plasma sample was added to 600 μL of acetonitrile and mixed. After 5 min of centrifugation at approximately 12,000 rpm (4 °C), the 500-μL supernatant was mixed with 30 mL of pyridine methoxyl amine (40 mg/mL) and incubated at 30 °C for 90 min for derivatization. Next, 100 μL of silicon alkylation reagents was added and the mixture was incubated at 40 °C for 30 min for derivatization. After another centrifugation for 5 min at approximately 12,000 rpm (4 °C), the supernatant was injected into the GC-MS system. The injection volume was 1 μL.
2.2. Experimental animals
2.7. GC-MS analysis
Female and male Wistar rats, weighing 130–150 g, were purchased from the pathogen-free facility at Beijing HFK Bioscience Technology Co. Ltd. (Beijing, China). The rats were housed in the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, and the experimental environment was maintained at 18–22 °C and a humidity of 55%–65%. The rats ate a standard diet and drank water ad libitum. All rats were allowed to adapt to the new environment for 7 days before drug administration. The protocols of the animal experiments were approved by the Laboratory Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (permit number: TCM-LAEC 2015012).
The MS conditions were as follows: ionization mode, EI; electron energy, 70eV; interface temperature, 250 °C; ion source temperature, 230 °C; transmission line temperature, 280 °C; quadrupole temperature, 150 °C; mass scan range, 60–600. The chromatographic column was HP5ms (30 m × 250 μm × 0.25 μm) and the carrier gas was He. The programmed temperature profile was 60 °C–325 °C at a rate of 10 °C/min, followed by maintenance for 10 min. The total analysis time was 37.5 min.
2. Materials and methods 2.1. Drug
2.8. GC-MS data processing The original data was imported into the AMDIS software (Agilent Technologies), and the data containing the deconvolution, integral, and metabolite identification were analyzed using the Fiehn 2013 Metabolomics RTL database. C8-28 fatty acid methyl ester was used to calibrate the retention time.
2.3. Subchronic toxicity treatment A total of 96 rats were randomly divided into four groups: control group, high-dose group (14 mg/kg), medium-dose group (7.0 mg/kg), and low-dose group (3.5 mg/kg), with equal numbers of male and female rats in each group. The rats in the three isopsoralen groups were intragastrically administered with isopsoralen once every day for 3 months, and the rats in the control group were treated with gum tragacanth solution. The general behavior of the rats was observed and recorded daily, and the weight, food consumption, and water consumption of the rats were detected weekly. Finally, the rats were anesthetized and then euthanized. Blood was collected to examine serum biochemical parameters, and the brain, heart, liver, spleen, kidney, adrenal glands, thymus, testis, epididymis, ovaries, and uterus were tested for visceral coefficients.
2.9. Metabolomic analysis Fold change analysis, principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and pathway analysis were performed using metaboanlayst 3.0 (http://www.metaboanalyst. ca/). The peak data were subjected to log transformation and Pareto scaling (mean-centered and divided by square root of standard deviation of each variable). The metabolites with significant changes induced by isopsoralen were evaluated as a VIP (variable importance in projection) of more than 1 and a P-value of less than 0.05 (t-test) between the control and isopsoralen groups. Finally, the biomarkers were interpreted using relevant biochemical databases, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG: http://www.kegg.com/).
2.4. Measurement of serum biochemical parameters Blood samples were collected, and placed into non-heparinized tubes for separation of serum. After 1 h, the solidified blood samples were centrifuged at 3500 rpm (10 min, 4 °C) and the supernatants were collected. Several serum biochemical parameters were detected by an
2.10. Statistical analysis The data were formulated as the mean ± SD for the tables and 2
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Fig. 1. Effects of repeated administration of isopsoralen on body weight in rats treated for 3 months. A) Female rats. B) Male rats. Data represent mean ± SEM.*P < 0.05, **P < 0.01, significant difference from control rats.
mean ± SEM for the figures. The significance of differences between the groups was evaluated by one-way analysis of variance (ANOVA) and Dunnett's t-test. Differences were considered statistically significant at P < 0.05.
Table 4 shows the serum biochemical parameters in male rats. Compared with the control group, TP activity was increased in all three groups treated with isopsoralen (P < 0.05 or P < 0.001). The levels of AST and BUN increased significantly in the 3.5 mg/kg and 14 mg/kg groups (P < 0.05, P < 0.01 or P < 0.001). In the 14 mg/kg group, the levels of TG, GLU, and CRE decreased significantly (P < 0.05), while the levels of TC, TBA, TG, and ALP in the 7 mg/kg dose group also decreased significantly (P < 0.05).
3. Results 3.1. Body weight, food intake and water intake After treatment with isopsoralen for 3 months, no rats died in the three dose groups. The color, appearance, and behavior of the rats were normal during the experimental period. The body weight of the rats in each group increased by varying degrees, and from week 7, the weights of the female rats treated with 14 mg/kg isopsoralen were lower than those in the control group (Fig. 1). Compared with the control group, the food and water consumption in the 14 mg/kg group decreased, but no significant difference was noted. The food and water consumption of mice in the isopsoralen groups showed no systematic variations (Fig. 2).
3.4. Histopathology In the present study, no abnormal gross pathology was observed during dissection. Some individual pathological changes were observed in the isopsoralen groups by microscopic examination. Compared with the control group, there were multiple points (more than 15) of necrosis or focal necrosis of hepatocytes (Fig. 3A) in the liver of the isopsoralen-treated rats, especially in the medium-dose and low-dose groups. In the pancreatic tissues, infiltration of inflammatory cells, mainly lymphocytes, was observed in the interstitium and arterioles of the pancreas (Fig. 3B) only in female rats in the 3.5 mg/kg (1/12) and 7.0 mg/kg (1/12) groups, while focal or lamellar vacuolar degeneration of the acinar (Fig. 3C) was observed in male rats in the 3.5 mg/kg (3/ 12) and 7.0 mg/kg (4/12) groups. In the male rat reproductive system, there were no appreciable changes in the control group, while the spermatogonium of the seminiferous tubule was degenerated, missing, or necrotic (Fig. 3D) in male rats in the 3.5 mg/kg (1/12) and 7.0 mg/kg (4/12) groups.
3.2. Visceral coefficients Table 1 shows the effects of isopsoralen on the visceral coefficients in female rats. Compared with the control group, the heart coefficient decreased significantly (P < 0.01) and the uterus coefficient increased significantly (P < 0.05) in the high-dose group (14 mg/kg). The ovary coefficient in the middle-dose group (7.0 mg/kg) increased significantly (P < 0.01). The thymus coefficient increased significantly (P < 0.05) and the adrenal gland coefficient also increased significantly (P < 0.05) in the low-dose group (3.5 mg/kg). Table 2 shows the effects of isopsoralen on the visceral coefficients in male rats. Compared with the control group, the testis coefficient in all groups treated with isopsoralen increased significantly (P < 0.05), and the heart coefficient in the 7.0 mg/kg group and the adrenal gland coefficient in the 14 mg/kg group also increased significantly (P < 0.05). No significant changes were observed in other visceral coefficients.
3.5. Metabolomic analyses in rat plasma Based on the m/z values and fragmentation patterns, 77 metabolites were identified in the GC-MS spectra of 24 plasma samples collected from the control and high-dose isopsoralen groups. The commonly used unsupervised multivariate statistical analysis (MVA) method PCA was used to examine the metabolomics data. The three-dimensional (3D) PCA score plots (Fig. 4A and B) indicated the dataset was of an acceptable quality and homogeneity without any outliers. Supervised PLS-DA was subsequently used to identify the differential features that were associated with the observed toxicities of isopsoralen on rats. An obvious separation between the female rats in the control and treated groups was observed in the PLS-DA score plot (Fig. 4C) with the class discrimination statistical parameters R2 of 0.9996 and Q2 of 0.6703. Variables contributing to the class clustering and discrimination were extracted via two criterions, including a value of the variable importance in the project (VIP) of more than 1 and p values from the ttest of less than 0.05. As a result, 19 metabolites were selected to
3.3. Blood biochemical parameters Several clinical parameters in serum were measured to monitor the toxic effects of isopsoralen. Table 3 shows the serum biochemical parameters in female rats. Serum GLU and CRE in all dose groups decreased significantly compared with the control group (P < 0.05 or P < 0.001). Other serum biochemical parameters were not significantly changed, except for a significant decrease in AST in the 3.5 mg/kg group (P < 0.01), and significant increases in ALP (P < 0.001) and BUN (P < 0.05). 3
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Fig. 2. Effects of repeated administration of isopsoralen on food and water intake in rats treated for 3 months. A) Food intake of female rats. B) Food intake of male rats.C) Water intake of female rats. D) Water intake of male rats. Data represent mean ± SEM.
Table 1 Effects of repeated administration of isopsoralen on visceral coefficients in female rats treated for 3 months (mean ± SD, n = 12). viscera coefficient
Control
brain heart thymus liver kidney adrenal glands spleen ovaries uterus
0.6074 0.3552 0.1257 2.5901 0.5862 0.0248 0.1805 0.0534 0.1821
± ± ± ± ± ± ± ± ±
0.0482 0.0308 0.0194 0.1442 0.0423 0.0028 0.0200 0.0087 0.0599
3.5 mg/kg
7.0 mg/kg
14 mg/kg
0.6149 0.3737 0.1487 2.6202 0.6075 0.0227 0.1728 0.0509 0.1802
0.6190 0.3450 0.1325 2.4920 0.6072 0.0229 0.1896 0.0617 0.1877
0.6317 0.3209 0.1222 2.4963 0.6173 0.0237 0.1782 0.0559 0.2598
± 0.0439 ± 0.0339 ± 0.0344* ± 0.2327 ± 0.0486 ± 0.0030* ± 0.0582 ± 0.0070 ± 0.0477
± 0.0396 ± 0.0284 ± 0.0276 ± 0.1087 ± 0.0403 ± 0.0029 ± 0.0395 ± 0.0068** ± 0.0647
± 0.0451 ± 0.0276** ± 0.0162 ± 0.0982 ± 0.0437 ± 0.0023 ± 0.0241 ± 0.0065 ± 0.1084*
*P < 0.05, **P < 0.01, significant difference from the control group. Table 2 Effects of repeated administration of isopsoralen on visceral coefficients in male rats treated for 3 months (mean ± SD, n = 12). viscera coefficient
Control
brain heart thymus liver kidney adrenal glands spleen testis epididymis
0.3969 0.3131 0.0881 2.2803 0.5626 0.0113 0.1706 0.7304 0.3050
± ± ± ± ± ± ± ± ±
0.0308 0.0277 0.0233 0.1526 0.0589 0.0021 0.0660 0.0550 0.0198
3.5 mg/kg
7.0 mg/kg
14 mg/kg
0.4122 0.3304 0.0999 2.4132 0.5880 0.0129 0.1739 0.8112 0.3094
0.4091 0.3454 0.1067 2.3367 0.5766 0.0130 0.1660 0.8028 0.2890
0.4071 0.3325 0.1024 2.3526 0.5698 0.0132 0.1558 0.8048 0.3053
± 0.0265 ± 0.0217 ± 0.0240 ± 0.3714 ± 0.0557 ± 0.0018 ± 0.0686 ± 0.0728* ± 0.0370
*P < 0.05, significant difference from the control group.
4
± 0.0361 ± 0.0295** ± 0.0197 ± 0.2032 ± 0.0400 ± 0.0021 ± 0.0334 ± 0.0695* ± 0.0397
± 0.0293 ± 0.0276 ± 0.0272 ± 0.1159 ± 0.0531 ± 0.0017* ± 0.0168 ± 0.0830* ± 0.0247
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Table 3 Effects of repeated administration of isopsoralen on blood biochemical parameters in female rats treated for 3 months (mean ± SD, n = 12). parameter
Control
3.5 mg/kg
7.0 mg/kg
14 mg/kg
ALT AST ALP TBIL TBA TG TC GLU BUN CRE CK TP ALB
26.18 ± 5.02 156.45 ± 28.73 49.36 ± 13.46 0.14 ± 0.03 70.12 ± 23.26 0.38 ± 0.11 1.91 ± 0.49 10.82 ± 2.25 5.18 ± 0.99 92.34 ± 8.76 1280.46 ± 1145.69 64.75 ± 2.83 28.37 ± 2.61
24.27 ± 5.78 131.18 ± 19.45* 81.55 ± 29.85*** 0.11 ± 0.04 82.83 ± 23.38 0.37 ± 0.15 2.03 ± 0.27 8.64 ± 2.19* 6.17 ± 1.25* 77.80 ± 4.95*** 771.73 ± 614.21 66.46 ± 3.43 27.59 ± 2.43
29.83 ± 14.80 148.67 ± 26.58 58.25 ± 20.04 0.18 ± 0.07 83.37 ± 40.64 0.43 ± 0.16 1.96 ± 0.30 7.18 ± 2.09*** 5.33 ± 1.02 61.61 ± 6.04*** 978.92 ± 419.41 65.95 ± 2.91 28.36 ± 2.19
23.80 ± 5.71 135.40 ± 21.20 50.00 ± 12.18 0.16 ± 0.06 80.52 ± 36.04 0.38 ± 0.15 2.19 ± 0.37 7.39 ± 1.64*** 5.04 ± 0.76 64.69 ± 4.72*** 1176.80 ± 800.81 66.80 ± 4.09 27.79 ± 1.51
*P < 0.05, **P < 0.01, ***P < 0.001, significant difference from the control group. Table 4 Effects of repeated administration of isopsoralen on blood biochemical parameters in male rats treated for 3 months (mean ± SD, n = 12). parameter
Control
3.5 mg/kg
7.0 mg/kg
14 mg/kg
ALT AST ALP TBIL TBA TG TC GLU BUN CRE CK TP ALB
42.67 ± 14.20 205.40 ± 52.41 107.90 ± 26.86 0.20 ± 0.08 126.86 ± 33.72 0.44 ± 0.17 1.60 ± 0.27 7.22 ± 1.99 5.92 ± 0.80 85.42 ± 10.88 2307.20 ± 1472.66 59.48 ± 1.63 23.56 ± 0.99
35.90 ± 13.00 146.22 ± 25.21*** 93.50 ± 16.92 0.15 ± 0.06* 119.51 ± 45.57 0.42 ± 0.11 1.69 ± 0.28 6.90 ± 3.37 5.10 ± 0.76* 79.08 ± 8.47 2023.20 ± 1333.42 62.77 ± 4.83* 23.82 ± 2.09
33.45 ± 11.92 203.00 ± 44.25 79.09 ± 14.27** 0.15 ± 0.05 87.82 ± 38.27* 0.30 ± 0.10* 1.31 ± 0.24* 6.79 ± 1.79 5.66 ± 0.69 81.89 ± 9.24 2403.27 ± 999.59 61.30 ± 1.69* 23.43 ± 1.34
35.64 ± 12.86 143.82 ± 20.50** 92.00 ± 23.14 0.16 ± 0.06 96.00 ± 50.89 0.29 ± 0.08* 1.46 ± 0.33 5.37 ± 1.10* 5.15 ± 0.61* 78.14 ± 6.46* 1335.91 ± 545.37* 64.13 ± 2.22*** 24.22 ± 0.80
*P < 0.05, **P < 0.01, ***P < 0.001, significant difference from the control group.
Fig. 3. Representative histopathological photographs of organ lesions in Wistar rats. A) Liver, with hepatocyte focal necrosis. B) Pancreas, with inflammatory cell infiltration.C) Pancreas, with vacuolar degeneration of the acinar. D) Testicle, with degeneration and necrosis of the spermatogonium. H&E staining, 200×.
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Fig. 4. PCA and PLS-DA score plots of plasma samples in the control and isopsoralen groups according to sex. A) PCA score plot of female rats. B) PCA score plot of male rats. C) PLS-DA score plot of female rats. D) PLS-DA score plot of male rats.
commonly affected by isopsoralenare intervention in both male and female rats, while the other metabolites were completely different. With an impact factor > 0.1 and raw p value < 0.1, the pathway topology and integrating enrichment analyses (Fig. 5B and C) showed that the metabolic changes induced by isopsoralen also had significant gender differences in rats. Aside from two common pathways (glycine, serine, and threonine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis), isopsoralen disturbed another six metabolism pathways (aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, pyruvate metabolism, alanine, aspartate and glutamate metabolism, D-glutamine and D-glutamate metabolism and linoleic acid metabolism) in female rats and four other pathways (glyoxylate and dicarboxylate metabolism, citrate cycle, glycerolipid metabolism, cysteine and methionine metabolism) in male rats.
indicate the impact of isopsoralen on the metabolism of female rats. Compared with the control group, the levels of pyruvic acid, L-lactic acid, 2-ketobutyric acid, oxalic acid, D-threitol, L-glutamic acid, fructose, L-lysine, tyrosine, and serotonin were up-regulated, while the levels of L-leucine, phosphoric acid, L-serine, palmitic acid, heptadecanoic acid, stearic acid, linoleic acid, alpha tocopherol, and cholesterol were down-regulated in the female administration group (Table 5). A PLS-DA model was also established to classify the samples from the control and treated male rats. The score plot (Fig. 4D) displayed a clustering trend for different groups with an R2 of 0.9980 and Q2 of 0.6505. Through the abovementioned criterions, 28 metabolites were extracted as the differential features for revealing the metabolic changes induced by isopsoralen on male rats. Compared with the control group, the content of indole 3-propionic acid was decreased in the male administration group, while the other 27 metabolites were increased (Table 6). Venn analysis (Fig. 5A) revealed that only three metabolites were 6
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2012a). Isopsoralen is a major active and quality-control component of Fructus Psoraleae in the Chinese pharmacopoeia and its pharmacological activities have been investigated. Interestingly, a study revealed that some compounds of Fructus Psoraleae were metabolized to isopsoralen by intestinal microflora through deglucosylation (Wang et al., 2014), indicating that the concentration of isopsoralen may be much higher in vivo. Therefore, aside from analyzing its historical and current applications, toxicity studies are urgently needed to assess the safety of the wide array of isopsoralen doses in use. Changes in body weight is the most basic indicator of toxicity toward organs and systems (Thinkratok et al., 2014; Santos et al., 2009). As shown in Fig. 1, from week 7 to the end of administration, the body weight gains in the 14 mg/kg dose group were significantly decreased in female rats compared with male rats. Furthermore, an increase in a visceral coefficient is an indication of cell swelling or inflammation, while a reduction in the same parameter can be attributed to cellular constriction (AOT Ashafa et al., 2009). In the present study, the heart coefficient of female rats in the isopsoralen groups was significantly lower than that in the control group, and the ovary coefficient was significantly higher. Additionally, the testis, heart, and adrenal gland coefficients of male rats were apparently increased compared with the control group. These findings imply that isopsoralen may have effects on the reproductive system in both female and male rats. Alterations in AST, ALP, and histopathologic features of the liver occurred in rats receiving treatment with isopsoralen. Serum levels of ALT, AST, and ALP are commonly used as markers of liver damage (Ozer et al., 2008). In our study, serum AST was significantly decreased and serum ALP was increased in female rats in the 3.5 mg/kg isopsoralen group, while serum AST and ALP were lower in male rats in all groups compared with the control rats. Additionally, the liver is the site of cholesterol disposal or degradation, is a major site of synthesis (Cofan Pujol, 2014), and it was observed that serum TC and TG were lower in male rats treated with isopsoralen compared with the control group. Decreased CRE in female rats was correlated with toxic changes in the kidney. Decreased GLU in female rats may be correlated with severe malnutrition, but we cannot make a conclusion with regards as to why the GLU decreased in this study. The BUN was increased in female rats treated with 14 mg/kg isopsoralen. In male rats, BUN was significantly decreased in the 3.5 and 14 mg/kg isopsoralen groups. Our results demonstrate that isopsoralen may have toxic effects compared with the control group. It is worth mentioning that such changes were not consistent over time or dose-related, and only appeared at the low dose. This phenomenon has been previously observed in some studies (Fagin, 2012), and researchers suggest that certain chemicals can have toxic effects at very low doses. To better observe the toxic effects of isopsoralen in rats, a metabolomic study of the biochemical profiles was performed by integrating GC-MS analyses. In our study, we found that the toxic effects of isopsoralen were sexdependent, with the hepatotoxicity and nephrotoxicity in female rats appearing to be more significant than those in male rats. Therefore, we performed PCA and PLS-DA to analyze and compare the metabolic profiles by sex. A total of 19 and 28 metabolites were significantly changed in the plasma samples from female rats and male rats, respectively. A metabolomic analysis is suitable for direct assessment of isopsoralen toxicity, and the biological relationships between the different metabolites and toxic effects induced by isopsoralen in both male and female rats are discussed. The disturbance of metabolic pathways primarily occurred in those involved in amino acid metabolism, regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Regarding phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine is converted to tyrosine in the liver by phenylalanine hydroxylase (Waters et al., 2000). An accumulation of phenylalanine in the blood leads to phenylketonuria (Al Hafid and Christodoulou, 2015). In our research, based on the findings of increased phenylalanine in male rats and decreased tyrosine in female rats (Fig. 6A), we propose that isopsoralen
Table 5 Significant alterations of plasma metabolites in female rats treated with isopsoralen (14 mg/kg). Metabolites
R.T.(min)
t- test
VIP
Trend
pyruvic acid L- lactic acid 2-Ketobutyric acid oxalic acid L-leucine phosphoric acid L-serine D-threitol L-glutamic acid fructose L-lysine tyrosine palmitic acid heptadecanoic acid stearic acid Linoleic acid serotonin alpha tocopher cholesterol
6.158 6.327 6.884 7.347 9.342 9.455 10.574 12.532 13.771 16.705 17.011 17.211 18.062 18.963 19.513 19.582 21.834 26.582 26.719
0.0013073 0.032428 0.048028 0.035542 0.044273 0.0035397 0.042941 0.0025924 0.023179 0.020118 0.011119 0.003015 0.026962 0.0029989 0.010864 0.029956 0.040543 0.034899 0.0007268
2.0809 1.3207 1.6059 1.3698 1.1499 1.9766 1.2278 1.6037 1.4477 1.511 1.7113 2.2409 1.1695 1.5662 1.3406 1.4944 1.7499 1.0666 1.4628
down down down down up up up down down down down down up up up up down up up
Table 6 Significant alterations of plasma metabolites in male rats treated with isopsoralen (14 mg/kg). Metabolites
R.T.(min)
t- test
VIP
Trend
indole 3-propionic acid pyruvic acid glycolic acid acetohydroxamic acid 2-hydroxybutyric acid oxalic acid N-methylalanine 2-ketoisocaproic acid ethanolamine caprylic acid glycerol phosphoric acid glycine glyceric acid fumaric acid tartronic acid L-methionine Beta-alanine D-malic acid phenylalanine L-pyroglutamic acid hypotaurine L-fucose O-phosphocolamine citric acid gluconic acid mucic acid myo-inositol
2.6142 6.158 6.521 7.09 7.284 7.347 7.922 8.523 9.261 9.28 9.405 9.455 9.824 10.187 10.293 10.38 11.137 11.4 12.22 12.851 12.601 13.539 15.128 15.654 16.048 17.655 18.143 18.806
0.025856 0.0013097 0.00085709 0.036681 0.0031334 0.02375 0.0022555 0.020824 0.0049312 0.0024793 0.0066685 1.88E-05 0.0027673 0.00010793 0.00061052 1.53E-05 0.0020704 0.0060127 0.00075731 0.011092 0.014067 0.006016 0.0062063 0.044344 0.00028363 0.0038772 0.0059397 0.0068841
1.173 1.6191 1.5738 1.2263 1.3283 1.2431 1.1775 1.1385 1.527 2.021 1.5146 1.3138 1.428 1.9922 1.7443 1.7673 1.3465 1.3106 1.7768 1.1014 1.375 1.0495 1.2324 1.1952 1.5757 1.6964 1.2756 1.225
down up up up up up up up up up up up up up up up up up up up up up up up up up up up
4. Discussion Traditional Chinese Medicine is widely used to cure and prevent various diseases, but there remain problems with its clinical use. Recently, the potential for adverse effects of Traditional Chinese Medicine in clinical usage has become an important issue (Lai et al., 2013). Fructus Psoraleae has been widely used for its therapeutic effects on osteoporosis, osteomalacia, bone fracture, vitiligo, and psoriasis. Nowadays, many clinical reports on adverse reactions of Fructus Psoraleae have caused social concern (Cheung et al., 2009). Toxicology studies on animals suggest that Fructus Psoraleae mainly exhibits toxicity toward the liver (Chitturi and Farrell, 2008; Wang et al.,
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Fig. 5. Pathway analyses for differential metabolites in plasma. A) Venn analysis of the metabolic biomarkers induced by isopsoralen on female and male rats; B) Bubble plot of the metabolism pathways disturbed by isopsoralen on female rats; C) Bubble plot of the metabolism pathways disturbed by isopsoralen on male rats. Fig. 6. Pathway analyses for plasma metabolites. A) phenylalanine, tyrosine, and tryptophan biosynthesis; B) glycine, serine and threonine metabolism. The metabolites with blue arrows were changed in male rats and the metabolites with red arrows were changed in female rats. (↑) indicates that the compound was upregulated in the model group and (↓) indicates that the compound was downregulated in the model group.
in mice, and Wang et al. (2016) studied urine metabolomics for the blood-replenishing mechanism of Angelica sinensis in a blood-deficient mouse model. These analyses may have ignored differential efficacies or toxicities based on sex. In fact, sex differences in human and animal toxicities have raised much concern (Gochfeld, 2007). Sex-dependent metabolism often leads to differences in pharmacological or toxicological responses to xenobiotics (Mugford and Kedderis, 1998). Sexdependent metabolic variations in Wistar rats were confirmed by Stanley et al. (2005) using 1H nuclear magnetic resonance spectroscopy of urine coupled with chemometric methods. In our study, we found that isopsoralen showed sex differences in its toxicities and plasma metabolomics. Compared with the control group, female rats were found to be more sensitive to isopsoralen than male rats, and the body weight gains of female rats in the 3.5 mg/kg dose group were
may affect the metabolic function of the liver. For glycine, serine, and threonine metabolism, glycine and serine have multiple physiological functions in animals and humans. Glycine is a major component of collagen and elastin, as the most abundant proteins in the body (Wu, 2009). Serine hydroxymethyltransferase catalyzes the formation of glycine from serine (Wang et al., 2013). In our research, markedly higher levels of glyceric acid and glycine were detected in male rats, as well as a markedly higher level of L-serine in female rats after isopsoralen treatment (Fig. 6B). Thus, isopsoralen may cause amino acid imbalances and toxicity. In many metabolism studies, the experiments were only performed on male animals. For example, Song et al. (2015) used GC/MS-based metabolomic profiling to study the therapeutic effects of a Huangbai–Zhimu herb-pair extract on streptozotocin-induced type 2 diabetes 8
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suppressed, especially during the last few weeks. Additionally, with respect to blood biochemical parameters, female rats appeared to be more sensitive to hepatotoxicity and nephrotoxicity than male rats after treatment with isopsoralen. Moreover, based on the analysis of metabolites, there were 19 metabolic differences in female rats and 28 metabolic differences in male rats after isopsoralen administration. As shown in Fig. 5, the primary affected pathways in female rats and male rats all included those involved in amino acid metabolism, but the potential biomarkers involved in the pathways were different (Fig. 6). Furthermore, fatty acid metabolism, such as linoleic acid metabolism and methane metabolism, were the main affected pathways in female rats, while lipid metabolism, such as glycerolipid metabolism, and energy metabolism, such as glyoxylate and dicarboxylate metabolism, were the main affected pathways in male rats. Thus, future studies should use balanced sexes and analyze data by sex and other interactions rather than simply ignoring the effect of sex. Further investigations are still needed to uncover the mechanisms of the sex differences in isopsoralen-induced toxicity.
683–685. Chitturi, S., Farrell, G.C., 2008. Hepatotoxic slimming aids and other herbal hepatotoxins. J. Gastroenterol. Hepatol. 23 (3), 366–373. Citarasu, T., et al., 2003. Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae. Aquacult. Int. : journal of the European Aquaculture Society 11, 583–595. Cofan Pujol, M., 2014. Mecanismos básicos. Absorción y excreción de colesterol y otros esteroles. Clín. Invest. Arterioscler. 26, 41–47. Fagin, D., 2012. Toxicology: the learning curve. Nature 490, 462–465. Feng, C.-Y., et al., 2014. Mitochondrial proteomic analysis of ecdysterone protection against oxidative damage in human lens epithelial cells. Int. J. Ophthalmol. 7, 38–43. Gochfeld, M., 2007. Framework for gender differences in human and animal toxicology. Environ. Res. 104, 4–21. Haraguchi, H., et al., 2002. Antioxidative components of Psoralea corylifolia (leguminosae) phytotherapy research volume 16, issue 6. Phytother Res. 16, 539–544. Khatune, N.A., et al., 2004. Antibacterial compounds from the seeds of Psoralea corylifolia. Fitoterapia 75, 228–230. Lai, J.-N., et al., 2013. Observational studies on evaluating the safety and adverse effects of traditional Chinese medicine. Evid. Based Complement Altern. Med. 2013, 1–9. Lim, S.-H., et al., 2009. Ethanol extract of Psoralea corylifolia L. and its main constituent, bakuchiol, reduce bone loss in ovariectomised Sprague-Dawley rats. Br. J. Nutr. 101, 1031–1039. Mar, W., et al., 2001. Cytotoxic constituents of Psoralea corylifolia. Arch Pharm. Res. (Seoul) 24, 211–213. Mugford, C.A., Kedderis, G.L., 1998. Sex-dependent metabolism of xenobiotics. Drug Metab. Rev. 30, 441–498. Nam, S.W., et al., 2005. A case of acute cholestatic hepatitis associated with the seeds of Psoralea corylifolia (Boh-Gol-Zhee). Clin. Toxicol. 43, 589–591. Nicholson, J.K., et al., 1999. 'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica; the fate of foreign compounds in biological systems 29, 1181–1189. Nicholson, J.K., Wilson, I.D., 2003. Understanding 'global' systems biology: metabonomics and the continuum of metabolism. Nat. Rev. Drug Discov. 2, 668–676. Ozer, J., et al., 2008. The current state of serum biomarkers of hepatotoxicity. Toxicology 245, 194–205. Powers, R., 2009. NMR metabolomics and drug discovery. Magn. Reson. Chem. 47, S2–S11. Santos, S.R., et al., 2009. Toxicological and phytochemical studies of Aspidosperma subincanum Mart. stem bark (Guatambu). Pharmazie 64, 836–839. Song, L., et al., 2015. Application of GC/MS-based metabonomic profiling in studying the therapeutic effects of Huangbai–Zhimu herb-pair (HZ) extract on streptozotocin-induced type 2 diabetes in mice. J. Chromatogr. B 997, 96–104. Stanley, E.G., et al., 2005. Sexual dimorphism in urinary metabolite profiles of Han Wistar rats revealed by nuclear-magnetic-resonance-based metabonomics. Anal. Biochem. 343, 195–202. Tang, S.Y., et al., 2004. Characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional Chinese medicines. Free Radic. Biol. Med. 36, 1575–1587. Thinkratok, A., et al., 2014. Safety assessment of hydroethanolic rambutan rind extract: acute and sub-chronic toxicity studies. Indian J. Exp. Biol. 52, 989–995. Tsai, M.-H., et al., 2007. Psoralea corylifolia extract ameliorates experimental osteoporosis in ovariectomized rats. Am. J. Chin. Med. 35, 669–680. Wang, J., et al., 2012a. Evaluation of hepatotoxicity and cholestasis in rats treated with EtOH extract of Fructus Psoraleae. J. Ethnopharmacol. 144, 73–81. Wang, T., et al., 2016. Urine metabonomic study for blood-replenishing mechanism of Angelica sinensis in a blood-deficient mouse model. Chin. J. Nat. Med. 14, 210–219. Wang, W., et al., 2013. Glycine metabolism in animals and humans: implications for nutrition and health. Amino Acids 45, 463–477. Wang, X., et al., 2012b. Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease. Mol. Cell. Proteomics : MCP. 11, 370–380. Wang, Y.-F., et al., 2014. A UPLC–MS/MS method for in vivo and in vitro pharmacokinetic studies of psoralenoside, isopsoralenoside, psoralen and isopsoralen from Psoralea corylifolia extract. J. Ethnopharmacol. 151, 609–617. Wang, Y., et al., 2011. Screening antitumor compounds psoralen and isopsoralen fromPsoralea corylifoliaL. Seeds. Evid. Based Complement Altern. Med. 2011, 1–7. Waters, P.J., et al., 2000. Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype. Mol. Genet. Metabol. 69, 101–110. Wu, G., 2009. Amino acids: metabolism, functions, and nutrition. Amino Acids 37, 1–17. Yuan, X., et al., 2016. Psoralen and isopsoralen ameliorate sex hormone deficiency-induced osteoporosis in female and male mice. BioMed Res. Int. 2016, 6869452. Zhang, A., et al., 2010. Metabolomics: towards understanding traditional Chinese medicine. Planta Med. 76, 2026–2035. Zhang, C.Z., et al., 2005. In vitro estrogenic activities of Chinese medicinal plants traditionally used for the management of menopausal symptoms. J. Ethnopharmacol. 98, 295–300.
5. Conclusions This study revealed the impact of isopsoralen by conducting subchronic toxicity experiments and employing GC/MS-based metabolomic profiling combined with multivariate statistical analysis. Visceral coefficients and serum biochemical parameters were significantly changed after isopsoralen administration. Female rats were more sensitive to isopsoralen-induced toxicity than male rats since the weights of the female rats significantly decreased and the infiltration of inflammatory cells was observed in the interstitium and arterioles of the pancreas only in female rats. The liver, pancreas, and reproductive system may be the target organs affected by isopsoralen based on the histopathological findings. The metabolic analysis of plasma samples revealed that isopsoralen disturbed the metabolism of amino acids. We also found that isopsoralen exhibited sex differences in its toxicities and plasma metabolomics. The mechanism of isopsoralen toxicity and its observed sex differences as identified by metabonomics in this study requires further research to confirm the pathways involved. Conflicts of interest The authors declare that there are no conflicts of interest in regards to this manuscript. Acknowledgments The work was supported by the National Natural Science Foundation of China (No.81202991, 816738261, 81703790). We thank Wei Nie and Hong Shi for their help in this work. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.yrtph.2019.01.010. References AI Hafid, N., Christodoulou, J., 2015. Phenylketonuria: a review of current and future treatments. Transl. Pediatr. 4, 304–317. AOT Ashafa, M.Y., Grierson, D.S., Afolayan, A.J., 2009. Toxicological evaluation of the aqueous extract of Felicia muricata Thunb. leaves in Wistar rats. Afr. J. Biotechnol. 8 (6), 949–954. Cheung, W.I., et al., 2009. Liver injury associated with the use of Fructus Psoraleae (Bolgol-zhee or Bu-gu-zhi) and its related proprietary medicine. Clin. Toxicol. 47,
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