Abstract

   Pre-clinical safety evaluation of traditional medicines is imperative
   because of the universality of drug-induced adverse reactions. Psoralen
   and isopsoralen are the major active molecules and quality-control
   components of a traditional herbal medicine which is popularly used in
   Asia, Fructus Psoraleae. The purpose of this study is to assess the
   long-term effects of psoralen and isopsoralen with low levels on the
   biochemical parameters and metabolic profiles of rats. Three doses (14,
   28, and 56 mg/kg) of psoralen and one dose (28 mg/kg) of isopsoralen
   were administered to rats over 12 weeks. Blood and selected tissue
   samples were collected and analyzed for hematology, serum biochemistry,
   and histopathology. Metabolic changes in serum samples were detected
   via proton nuclear magnetic resonance (^1H-NMR) spectroscopy. We found
   that psoralen significantly changed the visceral coefficients, blood
   biochemical parameters, and histopathology, and isopsoralen extra
   influenced the hematological index. Moreover, psoralen induced
   remarkable elevations of forvaline, isoleucine, isobutyrate, alanine,
   acetone, pyruvate, glutamine, citrate, unsaturated lipids, choline,
   creatine, phenylalanine, and 4-hydroxybenzoate, and significant
   reductions of ethanol and dimethyl sulfone. Isopsoralen only induced a
   few remarkable changes of metabolites. These results suggest that
   chronic exposure to low-level of psoralen causes a disturbance in
   alanine metabolism, glutamate metabolism, urea cycle, glucose-alanine
   cycle, ammonia recycling, glycine, and serine metabolism pathways.
   Psoralen and isopsoralen showed different toxicity characteristics to
   the rats.

   Keywords: Fructus Psoraleae, psoralen, isopsoralen, sd rats, toxicity,
   metabolomics

1. Introduction

   Fructus Psoraleae (F.P.), the seed of Psoralea corylifolia L.
   (Leguminosae), has been widely used in Asian countries as a traditional
   medicine with the effects of warming kidney [[34]1], activating “yang,”
   promoting inspiration [[35]2], and checking diarrhea [[36]3]. There are
   more than 40 prescriptions containing F.P. in the Chinese
   pharmacopoeia. However, with the continuously expanded clinical
   application of F.P., toxicity problems have been concerned by
   researchers and doctors. Yao et al. [[37]4] claimed that F.P. is one of
   the emerging hepatotoxins. Oral administration of the F.P. aqueous
   extract (25 g/kg) induced significantly liver and kidney injuries in
   Wistar rats, and the biomarkers related to glycerol phospholipids
   metabolism, amino acid metabolism, and energy metabolism, were largely
   changed [[38]5]. Psoralen (P) and isopsoralen (IP) are the major active
   molecules and quality-control components of F.P. [[39]6]. Many
   investigations have been conducted on the pharmacological properties of
   P and IP, such as antibacterial [[40]7], anti- inflammatory [[41]8],
   antitumor [[42]9], estrogen-like effect, and antioxidant activities
   [[43]10]. However, regardless of their therapeutic effects, high doses
   of P or IP are proved to be poisonous [[44]11,[45]12] and made major
   contribution to the toxicity induced by F.P. A study (in Chinese)
   [[46]13] about the toxicity intensity of F.P. prepared by different
   processing methods suggested that there is a positive correlation
   between the toxicity and content of P and IP. However, there lacks a
   systematic study of the toxicity, especially the long-term toxicity of
   P and IP. Few studies have been conducted on the mechanism of P or IP
   or their comparison.

   The traditional toxicity assessment method using biochemistry and
   histopathology lacks sensitivity and accuracy. Metabonomics [[47]14] is
   a relatively new research technique and has become a viable tool for
   investigating the biochemical effects of many toxic substances. It
   reflects the overall changes of the body through the metabolic changes
   of plasma or urine, during a genetic modification or physiological
   stimulus [[48]15]. With the continuous progress of the discipline,
   untargeted metabonomics has been widely used in drug safety assessment
   and toxicity prediction and provided valuable information for
   drug-induced cardiotoxicity, hepatotoxicity, and nephrotoxicity.

   In this study, the long-term toxicities induced by psoralen (P) and
   isopsoralen (IP) were performed in SD rats with a 12-week oral
   administration of P (14, 28, 56 mg/kg) or IP (28 mg/kg), and the
   reversibility, persistence, or delayed occurrence of target organ
   toxicity were detected after a 4-week recovery period. The results
   illustrated that chronic exposure to low-level of psoralen or
   isopsoralen injured the visceral coefficients, hematological and blood
   biochemical parameters, and histopathology of rats. Besides, psoralen
   caused a disturbance in alanine metabolism, glutamate metabolism, urea
   cycle, glucose-alanine cycle, ammonia recycling, glycine and serine
   metabolism pathways. Psoralen and isopsoralen showed different toxicity
   characteristics to the rats.

2. Results

2.1. Effects on the General Behavior and Mortality

   The administration of psoralen or isopsoralen for 12 weeks did not
   induce any behavioral modification at any time point in treated rats
   compared to their respective control group. The color, appearance, and
   behavior of the rats were normal and no death was recorded during the
   experimental period.

2.2. Effects on the Body Weight

   Body weight of each animal was measured every week throughout the
   study. [49]Figure 1 shows a graphical representation of average weights
   of rats during psoralen or isopsoralen administration and recovery
   periods. In all groups, body weights gradually increased from the 1st
   week to the 16th week, and body weights of animals in the treatment
   groups showed no significant decrease compared with that of control
   during 12 weeks of drug administration. However, the weights of the
   isopsoralen group were significantly lower than those of the control
   group ([50]Figure 1, P < 0.05) at the end of recovery period.

Figure 1.

   [51]Figure 1
   [52]Open in a new tab

   Weights of rats during 3 months of psoralen or isopsoralen
   administration and recovery period. Data represented as mean ± SD. * P
   < 0.05, significant difference from control rats.

2.3. Effects on the Food and Water Intakes

   [53]Figure 2A,B represent the effects of psoralen or isopsoralen on the
   food and water intakes in sub-acute treatment. It was noted that there
   was no consistent effects of food intake or water consumption by
   repeated oral administration of psoralen (14, 28 and 56 mg/kg) or
   isopsoralen (28 mg/kg) for SD rats in comparison with the control in
   the first 8 weeks. However, in the 9th week, the food intakes in the
   treated groups were significantly lower than those in the control group
   ([54]Figure 2A, P < 0.05 or P < 0.01). And the water consumptions in
   the psoralen (14, 28, and 56 mg/kg) and isopsoralen groups were
   significantly higher than those in the control group ([55]Figure 2B, P
   < 0.05, P < 0.01, or P < 0.001) from the 9th week to the end.

Figure 2.

   [56]Figure 2
   [57]Open in a new tab

   The rats’ food and water intake within 3 months’ repeated
   administration of psoralen or isopsoralen. (A) Food consumption. (B)
   Water consumption. Data represented as mean ± SD. * P < 0.05, ** P <
   0.01, *** P < 0.001, significant difference from control rats.

2.4. Effects on the Visceral Coefficients

   Organ-to-body weight ratio (relative organ weight) is a sensitive index
   for toxicity evaluation. The visceral coefficients of rats after 3
   months of repeated administration and 2 weeks of recovery are shown in
   [58]Table 1. Compared with the control group, the spleen coefficients
   in all groups treated with psoralen (14, 28, and 56 mg/kg) increased
   significantly (P < 0.05 or P < 0.01), and the thymus coefficients in
   the middle-dose group (28 mg/kg) and high-dose group (56 mg/kg)
   decreased significantly (all P < 0.01). Besides, the heart and adrenal
   gland coefficients significantly increased (all P < 0.05), and the
   liver coefficient significantly decreased (P < 0.01) in the psoralen
   low-dose group (14 mg/kg). In the psoralen middle-dose group (28
   mg/kg), the brain coefficient increased significantly (P < 0.05), while
   the liver coefficient significantly decreased (P < 0.05). Moreover,
   daily administration of isopsoralen (28 mg/kg) significantly increased
   the coefficients of brain and heart (P < 0.05 and P < 0.001). However,
   in the recovery period, only a significant increase (P < 0.05) in liver
   coefficient of the psoralen low-dose group (14 mg/kg) and a decrease (P
   < 0.05) in ovary coefficient of the psoralen high-dose group (56 mg/kg)
   were observed. No statistically significant difference was detected in
   other visceral coefficients.

Table 1.

   Visceral coefficients of rats after 3 months of repeated administration
   and 4 weeks of recovery, respectively (mean ± SD, n = 12 for
   administration and n = 6 for recovery).
   Organs Dose Brain Heart Liver Thymus Spleen Kidney Adrenal Gland Ovary
   Uterus
   12 weeks Control 0.648 ± 0.064 0.295 ± 0.023 3.000 ± 0.202 0.150 ±
   0.025 0.180 ± 0.020 0.675 ± 0.062 0.026 ± 0.005 0.063 ± 0.011 0.175 ±
   0.036
   P14 mg/kg 0.687 ± 0.044 0.320 ± 0.028 * 2.653 ± 0.207 ** 0.140 ± 0.026
   0.216 ± 0.041 * 0.673 ± 0.040 0.031 ± 0.003 * 0.066 ± 0.007 0.155 ±
   0.028
   P28 mg/kg 0.710 ± 0.050 * 0.325 ± 0.030 2.731 ± 0.318 * 0.120 ± 0.028
   ** 0.211 ± 0.027 ** 0.714 ± 0.060 0.029 ± 0.006 0.070 ± 0.015 0.160 ±
   0.036
   P56 mg/kg 0.685 ± 0.046 0.307 ± 0.021 2.876 ± 0.181 0.121 ± 0.026 **
   0.230 ± 0.040 ** 0.672 ± 0.029 0.027 ± 0.005 0.064 ± 0.010 0.183 ±
   0.068
   IP28 mg/kg 0.696 ± 0.045 * 0.348 ± 0.028 *** 2.793 ± 0.368 0.149 ±
   0.046 0.209 ± 0.051 0.696 ± 0.043 0.028 ± 0.006 0.065 ± 0.009 0.153 ±
   0.031
   Recovery Control 0.575 ± 0.040 0.335 ± 0.020 2.584 ± 0.230 0.168 ±
   0.018 0.180 ± 0.021 0.603 ± 0.046 0.021 ± 0.005 0.050 ± 0.004 0.192 ±
   0.075
   P14 mg/kg 0.590 ± 0.043 0.355 ± 0.020 2.894 ± 0.131 * 0.157 ± 0.016
   0.178 ± 0.021 0.557 ± 0.153 0.021 ± 0.004 0.048 ± 0.009 0.162 ± 0.022
   P28 mg/kg 0.583 ± 0.077 0.354 ± 0.020 2.781 ± 0.137 0.182 ± 0.031 0.192
   ± 0.020 0.618 ± 0.029 0.020 ± 0.004 0.045 ± 0.007 0.147 ± 0.015
   P56 mg/kg 0.567 ± 0.034 0.334 ± 0.015 2.592 ± 0.138 0.153 ± 0.021 0.186
   ± 0.014 0.624 ± 0.038 0.023 ± 0.002 0.044 ± 0.003 * 0.227 ± 0.111
   IP28 mg/kg 0.605 ± 0.048 0.350 ± 0.028 2.491 ± 0.194 0.153 ± 0.017
   0.198 ± 0.018 0.593 ± 0.021 0.024 ± 0.004 0.048 ± 0.006 0.179 ± 0.090
   [59]Open in a new tab

   * P < 0.05, ** P < 0.01, *** P < 0.001, significant difference from the
   control group.

2.5. Effects on Hematological Parameters

   The hematopoietic system is one of the most sensitive targets for toxic
   chemicals and served as an important index of overall health status for
   both humans and animals [[60]16]. The hematological parameters of the
   experimental and control rats are presented in [61]Table 2. There were
   no significant changes observed in the psoralen middle-dose (28 mg/kg)
   and high-dose (56 mg/kg) groups when compared with the control group (P
   > 0.05), although a significant change was found in white blood cell
   (WBC) in the psoralen low-dose (14 mg/kg) group. More importantly, in
   the isopsoralen group (28 mg/kg), there were significant increase in
   the percentage of white blood cells (WBC) (P < 0.05) and decreases in
   hemoglobin (HGB), mean corpuscular HGB (MCH), and mean corpuscular HGB
   concentration (MCHC) (all P < 0.001). After the recovery period, all
   the changed parameters in the isopsoralen group were returned to the
   range of normal physiological variations compared to the control (P >
   0.05).

Table 2.

   The blood biochemical parameters of rats after 3 months of repeated
   administration and 2 weeks of recovery, respectively (mean ± SD, n = 12
   for administration and n = 6 for recovery).
   Parameters ALT AST ALP TBIL TC TG TP ALB BUN CRE CK GLU
   12 weeks Control 29.00 ± 5.59 121.09 ± 19.11 150.36 ± 52.87 0.23 ± 0.10
   1.00 ± 0.17 0.84 ± 0.20 56.48 ± 7.46 25.87 ± 1.68 5.95 ± 2.03 69.71 ±
   11.18 968.55 ± 373.69 13.86 ± 1.28
   P14 mg/kg 27.92 ± 8.34 116.75 ± 11.96 54.00 ± 12.38 *** 0.22 ± 0.07
   1.18 ± 0.26 0.50 ± 0.09 *** 54.54 ± 2.01 23.34 ± 0.96 *** 3.26 ± 0.69
   *** 66.13 ± 3.12 375.67 ± 110.47 *** 11.68 ± 2.47 *
   P28 mg/kg 26.25 ± 8.55 130.67 ± 38.00 46.25 ± 9.92 *** 0.30 ± 0.12 1.02
   ± 0.42 0.86 ± 0.43 55.81 ± 3.25 24.12 ± 1.13 ** 3.85 ± 0.67 ** 66.58 ±
   5.56 420.92 ± 210.98 *** 8.48 ± 2.24 ***
   P56 mg/kg 25.00 ± 4.61 115.92 ± 26.82 55.83 ± 14.86 *** 0.28 ± 0.11
   1.01 ± 0.39 0.84 ± 0.40 54.99 ± 2.52 24.47 ± 1.46 * 3.21 ± 0.62 ***
   67.50 ± 4.12 535.58 ± 209.83 ** 11.67 ± 1.39 ***
   IP28 mg/kg 27.00 ± 5.95 109.83 ± 25.96 50.17 ± 9.19 *** 0.22 ± 0.08
   1.27 ± 0.41 0.68 ± 0.19 55.71 ± 1.99 24.32 ± 1.25 * 3.59 ± 0.6 ***
   66.39 ± 8.78 518.33 ± 246.45 ** 10.39 ± 3.94 *
   Recovery Control 36.33 ± 5.92 97.33 ± 11.33 71.33 ± 30.03 0.23 ± 0.09
   1.54 ± 0.28 0.61 ± 0.36 59.45 ± 2.70 26.27 ± 1.44 3.81 ± 0.22 68.20 ±
   2.05 564.83 ± 166.87 12.53 ± 2.81
   P14 mg/kg 37.33 ± 3.67 108.17 ± 7.76 88.83 ± 29.55 0.18 ± 0.06 1.49 ±
   0.15 1.31 ± 0.83 61.00 ± 3.48 27.41 ± 1.84 4.50 ± 0.47 ** 68.58 ± 2.63
   895.00 ± 342.46 13.56 ± 2.70
   P28 mg/kg 38.50 ± 4.23 104.50 ± 17.74 60.33 ± 7.94 0.23 ± 0.09 1.58 ±
   0.25 0.62 ± 0.22 60.40 ± 0.85 26.72 ± 0.87 3.86 ± 0.51 69.09 ± 4.47
   689.83 ± 206.08 13.83 ± 0.92
   P56 mg/kg 38.00 ± 4.60 114.33 ± 11.33 * 91.17 ± 29.26 0.21 ± 0.05 1.23
   ± 0.18 * 0.55 ± 0.18 59.49 ± 1.84 26.25 ± 1.49 4.50 ± 0.49 * 67.44 ±
   3.32 840.83 ± 442.98 13.61 ± 1.94
   IP28 mg/kg 34.40 ± 3.13 89.60 ± 7.89 60.80 ± 14.81 0.23 ± 0.07 1.49 ±
   0.41 0.44 ± 0.13 59.56 ± 1.65 25.91 ± 1.47 3.62 ± 0.54 67.09 ± 3.65
   814.40 ± 550.44 12.46 ± 2.13
   [62]Open in a new tab

   * P < 0.05, ** P < 0.01, *** P < 0.001, significant difference from the
   control group.

2.6. Effects on Blood Biochemical Parameters

   The effects of repeated administration of psoralen or isopsoralen and
   recovery in rats on serum biochemical parameters are summarized in
   [63]Table 3. All doses of psoralen (14, 28 and 56 mg/kg) and
   isopsoralen (28 mg/kg) treatment significantly decreased the relative
   levels of ALP, ALB, BUN, CK, and GLU ([64]Table 3, P < 0.05, P < 0.01
   or P < 0.001) in comparison with the control group. Furthermore,
   low-dose of psoralen (14 mg/kg) reduced the TG level at 12th-week
   administration (P < 0.05). No statistically significant differences
   were observed among the levels of ALT, AST, TBIL, TC, TP, or CRE
   between the control and treated animals (P > 0.05). However, in the
   recovery period, all the damage was abolished except for increases of
   AST in the psoralen low-dose (14 mg/kg) group (P < 0.05) and BUN in the
   psoralen low-dose (14 mg/kg) and middle-dose (28 mg/kg) groups (P <
   0.01 and P < 0.05, respectively).

Table 3.

   Hematological values of rats after 3 months of repeated administration
   and 2 weeks of recovery, respectively (mean ± SD, n = 12 for
   administration and n = 6 for recovery).
   Parameters WBC (10^9/L) RBC (10^12/L) HGB (g/L) MCV (fl%) MCH (pg) MCHC
   (g/L) PLT (10^10/L)
   12 weeks Control 1.88 ± 1.14 4.34 ± 1.93 85.42 ± 37.08 54.67 ± 2.50
   19.83 ± 0.73 362.67 ± 13.55 421.67 ± 233.20
   P14 mg/kg 4.07 ± 2.66 * 5.59 ± 1.47 111.08 ± 24.58 53.74 ± 2.90 20.08 ±
   1.25 373.92 ± 12.84 * 650.58 ± 346.52
   P28 mg/kg 2.98 ± 4.77 3.96 ± 2.10 76.25 ± 41.05 53.55 ± 3.04 19.34 ±
   1.01 361.67 ± 14.39 430.33 ± 425.39
   P56 mg/kg 2.72 ± 2.09 4.10 ± 2.36 72.00 ± 48.09 55.04 ± 4.31 16.84 ±
   6.47 304.00 ± 112.82 515.83 ± 396.29
   IP28 mg/kg 3.53 ± 2.38 * 5.11 ± 1.94 38.58 ± 17.45 *** 54.10 ± 3.54
   7.34 ± 2.60 *** 135.00 ± 45.26 *** 620.17 ± 423.04
   Recovery Control 3.02 ± 0.75 6.91 ± 0.51 138.67 ± 3.39 53.42 ± 2.61
   20.15 ± 1.36 377.17 ± 7.73 959.83 ± 75.28
   P14 mg/kg 2.73 ± 0.87 6.44 ± 1.24 128.33 ± 26.17 53.42 ± 1.95 19.88 ±
   0.65 372.00 ± 7.75 913.33 ± 193.37
   P28 mg/kg 2.35 ± 0.40 6.68 ± 1.19 130.52 ± 2.97 53.25 ± 1.47 19.55 ±
   0.53 367.33 ± 9.79 970.17 ± 274.90
   P56 mg/kg 2.77 ± 0.59 7.08 ± 1.68 137.00 ± 31.10 53.00 ± 1.75 19.4 ±
   50.85 366.67 ± 6.09 * 737.33 ± 313.05
   IP28 mg/kg 2.23 ± 0.99 7.98 ± 0.89 * 159.17 ± 18.05 * 53.22 ± 1.57
   19.95 ± 0.63 375.00 ± 9.19 678.05 ± 414.28
   [65]Open in a new tab

   * P < 0.05, ** P < 0.01, *** P < 0.001, significant difference from the
   control group.

2.7. Effects on the Histopathology of the Major Organs

   Histopathological examination of the major organs is presented in
   [66]Figure 3. Some individual pathological changes were observed in the
   psoralen high-dose (56 mg/kg) group and isopsoralen (28 mg/kg) group.
   The major pathological findings from the histopathological examination
   included portal myocardial inflammatory cell infiltration in the heart,
   minimal inflammatory cell foci, and vacuolar degeneration in the liver,
   and distal convoluted tubule cortical vacuolar degeneration and protein
   cast in the kidneys ([67]Figure 3). There was no obvious pathological
   change in the animals of recovery period.

Figure 3.

   Figure 3
   [68]Open in a new tab

   Histopathological photographs of organ lesions in Sprague–Dawley (SD)
   rats. H & E staining showing the heart, liver, and kidney injuries
   induced by psoralen and isopsoralen, 100× and 200×.

2.8. Metabolites Present in Serum Samples

   Typical ^1H-NMR spectrum of rat serum sample is shown in [69]Figure 4
   and the proton signals were assigned to 42 metabolites according to the
   literatures, standard references, public databases, and information