The role of hepatic antioxidant capacity and hepatobiliary transporter in liver injury induced by isopsoralen in zebrafish larvae

Abstract

Isopsoralen is the main component of the Chinese medicine psoralen, which has antitumour activity and can be used for the treatment of osteoporosis. However, the mechanism behind its hepatotoxicity has not yet been elucidated. In this study, the hepatotoxicity of isopsoralen was investigated using zebrafish. Isopsoralen treatment groups of 25, 50 and 100 μM were established. The mortality, liver morphology changes, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver histopathology and mRNA levels of liver injury–related genes in zebrafish larvae were measured. The results showed that isopsoralen resulted in the development of malformed zebrafish, dose-dependent increases in ALT and AST, decreased liver fluorescence and weakened fluorescence intensity. Histopathological examination showed that high-dose isopsoralen caused a large number of vacuolated structures in the larvae liver. The polymerase chain reaction results showed a significant decrease in the mRNA levels of genes related to antioxidant capacity (lfabp, gstp2 and sod1) and drug transport (mdr1, mrp1 and mrp2), indicating that isopsoralen significantly inhibited liver antioxidant capacity and drug efflux capacity in zebrafish larvae. Isopsoralen is hepatotoxic to zebrafish larvae via inhibition of drug transporter expression resulting in the accumulation of isopsoralen in the body and decreased antioxidant capacity, leading to liver injury.

Keywords

Isopsoralen hepatotoxicity zebrafish antioxidant capacity hepatobiliary transporter

Introduction

Psoralen, also known as Fructus Psoraleae, is the dry mature fruit of the leguminous plant psoralen (Psoralea corylifolia L.) and can function as an antitumour agent, osteoporosis and vitiligo treatment, sex hormone-like drug, immune booster, antidepressant and antibacterial agent.¹ However, there are increasing reports of liver injury caused by the use of psoralen and its related agents.^2,3 Isopsoralen is a coumarin compound isolated from psoralen, which is the main component of psoralen⁴ and is one of the quality control components of psoralen in the ‘Pharmacopoeia of the People’s Republic of China’ (2015 version). It has been reported that isopsoralen is likely to mediate the liver injury caused by psoralen.⁵ Researchers have investigated the manifestation and mechanism of isopsoralen-induced liver injury. For example, Wang et al. found that continuous intragastric administration of isopsoralen at 40 mg/kg for 28 days caused liver injury in mice, but the mechanism of hepatotoxicity has not yet been elucidated.⁵

As an ideal vertebrate model, zebrafish have unique advantages for evaluating drug toxicity, with a short reproductive cycle, in vitro embryo development, overall embryo transparency, good species stability, small individual differences, easy maintenance and low cost. Zebrafish have been widely used in toxicology research including developmental toxicology, environmental toxicology and drug toxicology.^6,7 A study by Chu et al. showed that in zebrafish, liver morphology was initially formed in the hours after birth and that the liver can rapidly grow in 60–72 h after birth until it reaches the appropriate size.⁸ At the molecular level, the molecular mechanism of zebrafish liver development is consistent with that of mammals, and its hepatocellular function and histopathological changes in a variety of liver diseases are also very similar to those of humans.⁹

Therefore, in this study, the liver fluorescent transgenic zebrafish at 72-h post-fertilization (hpf) was used as a model for liver toxicity evaluation to investigate the hepatotoxicity and toxicity mechanisms of isopsoralen, which is expected to provide the experimental support for the subsequent whole animal experiments and elucidation of the hepatotoxicity mechanism of isopsoralen at a molecular level.

Materials and methods

Chemicals

Isopsoralen, a yellow flocculent powder, was prepared and provided by the Binhai Laboratory, Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, China (with a purity greater than 98% as determined by high performance liquid chromatography (HPLC)). Isopsoralen was dissolved in dimethyl subfamily (dimethyl sulfoxide or DMSO) to prepare the stock solution. The samples were stored at 4°C and diluted with zebrafish embryo medium (5 mM sodium chloride, 0.17 mM potassium chloride, 0.4 mM calcium chloride and 0.16 mM magnesium sulfate) to obtain the desired concentration.

Zebrafish

In this study, liver fluorescent transgenic zebrafish (liver-fatty acid binding protein (L-FABP): enhanced green fluorescent protein (EGFP)) were used. The male and female zebrafish were separately maintained under the standard conditions of a light–dark 14 h:10 h lighting cycle at 28°C with regular feeding of granular bait and artemia. To obtain the eggs, healthy and mature zebrafish were placed into a mating cylinder at a male to female ratio of 1:1 or 1:2, and the fertilized eggs were collected at 9:00–10:00 a.m. the following day. After the fertilized eggs were sterilized and washed, they were transferred into the zebrafish embryo culture medium and cultured at 28°C with lighting control. In the drug treatment experimental groups, the 72-hpf larvae were treated with the embryo culture medium containing different concentrations of isopsoralen. All experimental procedures were conducted in accordance with the standard ethical guidelines and under control of the Biology Institute of Shandong Academy of Sciences ethical committee.

Drug treatment

When the zebrafish development reached 72 hpf, the normally developed larvae were selected and transferred into the 6-well plates with 10 larvae in each well. According to the pre-experiment results, isopsoralen treatment groups of 25, 50 and 100 μM along with a no treatment control group (zebrafish were maintained in the embryo culture medium containing 0.5% DMSO) were set up with three duplicate wells for each group. Subsequently, the zebrafish were incubated at a constant temperature (28°C) in a light incubator with the appropriate treatment conditions for three consecutive days, and the medium was changed daily.

Mortality rate of the zebrafish larvae

The mortality of the larvae in each experimental group was observed and recorded at 24, 48 and 72 h post-exposure (hpe; larvae survival was judged by the existence of a heartbeat).

Morphological changes in the zebrafish larvae

At 24, 48 and 72 hpe, the zebrafish were anesthetized by tricaine at a mass concentration of 0.3% for 1 min and then fixed on a slide with 3% methylcellulose. Images were then acquired on the side with a fixed focal length. The morphological changes in the larvae were observed using a fluorescence microscope. Zebrafish teratogenicity was scored according to previously described methods.¹⁰ The length of each group of larvae was measured using the software Image-Pro Plus 5.1 (the length from the head to the tip of the tail along the length of the larva was measured).

The effects of isopsoralen at different concentrations and different time points on the teratogenicity scores of the zebrafish were evaluated. The scoring standards were as follows: 5 – normal structure; 4 – slight change in the normal structure, suggesting a certain developmental delay or a restorable change; 3 – slight structural abnormality, usually showing only one deformity; 2 – moderate structural deformity, usually showing two or more deformities; 1 – severe structural malformation, usually showing a variety of deformities at the same time; and 0.5 – lack of structure.

Changes to liver fluorescence in zebrafish larvae

At 24, 48 and 72 hpe, the zebrafish were anesthetized with tricaine at a mass concentration of 0.3% for 1 min, and zebrafish images were captured from a fixed position on the side (two eyes overlapping). The liver fluorescence of zebrafish was recorded by fluorescence microscopy.

Determination of transaminase levels in zebrafish larvae liver

At 72 hpe, zebrafish larvae tissue homogenate was prepared to detect tissue levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

Histopathological examination of zebrafish larvae liver tissue

At 72 hpe, the zebrafish larvae were fixed in 4% paraformaldehyde fixing solution. The samples were dehydrated with gradient ethanol, cleared with xylene, embedded in paraffin, sliced, stained with haematoxylin and eosin (HE) and mounted. The slides were then observed under an optical microscope.

The mRNA levels of genes related to liver injury in zebrafish larvae were determined by quantitative real-time polymerase chain reaction

At 72 hpe, 15 zebrafish larvae per group were collected and stored in liquid nitrogen for quantitative real-time polymerase chain reaction (PCR). The liquid nitrogen–preserved zebrafish larvae tissues were carefully thawed and placed in a homogenizer. The RNA was extracted from the zebrafish tissue at 72 hpe using NanoMag Animal and Fish RNA Isolation Kit (Shannuo Scientific Company, Tianjin, China). Reverse transcription was performed for the RNA samples of each group to obtain the cDNA, and the expression levels of genes related to liver injury were measured using a Bio-RAD (CA, USA) TQ5 Multi-Colour Real-Time PCR Detection System.

The conditions for real-time quantitative PCR amplification were as follows: 1 cycle of pre-denaturation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 10 s; annealing at 60°C for 10 s; and extension at 65°C for 10 s for a total of 41 cycles. The fluorescence signal was recorded after the annealing cycle. The relative quantitative analysis of the results was performed using β-actin as the internal reference. The PCR primers of the target genes and the internal reference gene β-actin were synthesized by Nanjing Shengxing Biotechnology Co., Ltd, China. The designed gene primers are shown in Table 1.

Table 1.

Primers for real-time PCR.

Gene	Primer	Neucleotide sequence
β-actin	Forward	5′-AGAGCTATGAGCTGCCTGACG-3′
β-actin	Reverse	5′-CCGCAAGATTCCATACCCA-3′
lfabp	Forward	5′-ACGTGGCAGGTTTACGCTCAG-3′
lfabp	Reverse	5′-TTGGAGGTGATGGTGAAGTCG-3′
gstp2	Forward	5′-CACAGACCTCGCTTTTCACAC-3′
gstp2	Reverse	5′-GAGAGAAGCCTCACAGTCGT-3′
sod1	Forward	5′-GGCCAACCGATAGTGTTAGA-3′
sod1	Reverse	5′-CCAGCGTTGCCAGTTTTTAG-3′
mdr1	Forward	5′-AAAGCACAAAGGCAAAAAGG-3′
mdr1	Reverse	5′-TGTCAAACCACGACATCTGC-3′
mrp1	Forward	5′-AGGCCTGTGCTCTTCTACCA-3′
mrp1	Reverse	5′-AGAGAGCGGGTCGTCTAACA-3′
mrp2	Forward	5′-TCAGCGAGTACACGGAGATG-3′
mrp2	Reverse	5′-GCACCTGTTCGACCCACTAT-3′

PCR: polymerase chain reaction.

Statistical methods

The experimental data are presented as mean ± standard error, and the statistical differences were analyzed using one-way analysis of variance and Dunnett’s t-test. Differences with p < 0.05 were considered statistically significant, and differences with p < 0.01 were considered extremely statistically significant.

Results

Effects of isopsoralen on zebrafish larvae mortality

The survival of the zebrafish larvae was observed at 24, 48 and 72 h after treatment with different concentrations of isopsoralen. With the increase in drug concentration and treatment time, zebrafish mortality also increased. The results are shown in Table 2.

Table 2.

Cumulative mortality of zebrafish larvae after 24, 48 and 72 h exposure to isopsoralen doses (mean ± SE, n = 60).

Group	Mortality^a (%)
Group	24 hpe	48 hpe	72 hpe
Control	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Isopsoralen 25 μM	3.33 ± 1.67	5.00 ± 0.00	8.33 ± 1.67^b
Isopsoralen 50 μM	5.00 ± 2.89	13.33 ± 1.67^b	20.00 ± 2.89^b
Isopsoralen 100 μM	11.67 ± 4.41	15.00 ± 5.00^c	30.00 ± 2.89^b

SE: standard error; hpe: hours post-exposure.

^aThe data were obtained from 3 independent experiments.

^b P < 0.01 versus control.

^c P < 0.05 versus control.

Effects of isopsoralen on zebrafish larvae morphology

The experimental results showed that isopsoralen could cause development malformations in zebrafish larvae, manifesting as reduced or missing swim bladders (indicated by the red arrow) and a delay in yolk sac absorption (indicated by the yellow arrow), as shown in Figure 1(a) to (c). The scoring results showed that the zebrafish morphology score decreased with increasing drug concentration and prolonged treatment, which further confirmed the toxic effect of isopsoralen on zebrafish larvae development (Figure 1(d) to (f)). At 24, 48 and 72 h after drug exposure, the lengths of the zebrafish in the experimental groups were significantly shorter than those of the no treatment control group (Figure 2).

Figure 1.

Effect of isopsoralen on the developmental toxicity of zebrafish larvae at 24, 48 and 72 h after the drug exposure. (a) to (c) Effects of isopsoralen on zebrafish larvae morphology. (d) to (f) Statistical analysis graph of zebrafish morphological development scores.

Figure 2.

Effect of isopsoralen on the length of the zebrafish larvae at 24, 48 and 72 h after drug exposure. *p < 0.05, **p < 0.01 versus control.

Effects of isopsoralen on hepatotoxicity in zebrafish larvae

Fluorescence microscopy observation revealed that compared with the no treatment control group, the liver fluorescence area and the fluorescence intensity of the zebrafish larvae at 48 h after the administration of 100 μM isopsoralen and at 72 h after the administration of 50 and 100 μM isopsoralen were significantly decreased, indicating that isopsoralen caused obvious liver atrophy in the zebrafish, as shown in Figure 3.

Figure 3.

Effect of isopsoralen on zebrafish larvae liver at 24, 48 and 72 h after the drug treatment. (a) Effect of isopsoralen on fluorescent-labelled zebrafish liver. (b) Effect of isopsoralen on the fluorescence area of zebrafish liver. (c) Effect of isopsoralen on the fluorescence intensity of zebrafish liver. *p < 0.05, **p < 0.01 versus control.

Effects of isopsoralen on liver transaminase activity in zebrafish larvae

Compared with the no treatment control group, at 72 h after isopsoralen exposure in the zebrafish larvae, the ALT of the zebrafish larvae tissue was significantly increased in the 50 and 100 μM isopsoralen treatment groups (p < 0.05, p < 0.01; Figure 4), and the AST of the zebrafish larvae tissue was significantly increased in the 100 μM isopsoralen treatment group (p < 0.01; Figure 4).

Figure 4.

Effect of isopsoralen on ALT and AST levels in the zebrafish larvae. *p < 0.05, **p < 0.01 versus control. ALT: alanine aminotransferase; AST: aspartate aminotransferase.

Observation of the histopathological changes in zebrafish liver by HE staining

The results showed that the liver structures of normal zebrafish larvae were intact, and hepatocyte cytoplasm was evenly stained red. At 72 h after isopsoralen treatment, zebrafish hepatocytes in the 25 and 50 μM groups showed no significant changes. However, the hepatocytes of the zebrafish in the 100 μM treatment group were significantly decreased and had loose cytoplasm and large numbers of vacuoles (Figure 5).

Figure 5.

Effects of isopsoralen treatment on zebrafish liver tissue at 72 h after the drug treatment. (a) Control, (b) 25 μM isopsoralen, (c) 50 μM isopsoralen and (d) 100 μM isopsoralen. Loose cytoplasm is indicated by black solid arrowheads and vacuole is indicated by black dotted arrowheads.

Effect of isopsoralen on the mRNA expression levels of the genes related to liver injury in zebrafish larvae

Compared with the control group, at 72 h after treatment with isopsoralen, the mRNA expression levels of sod1 and mrp2 in the zebrafish in the 25 μM group were significantly decreased; the mRNA expression levels of lfabp, gstp2, sod1, mdr1 and mrp2 of the zebrafish in the 50 μM group were significantly decreased; and the mRNA expression levels of lfabp, gstp2, sod1, mdr1, mrp1 and mrp2 of the zebrafish in the 100 μM group were significantly decreased (Figure 6).

Figure 6.

Gene expression levels of lfabp, gstp2, sod1, mdr1, mrp1 and mrp2 in zebrafish embryos exposed to 0, 25, 50 or 100 µM isopsoralen at 72 hpe. The mRNA expression levels are represented as fold-changes relative to the control group. *p < 0.05, **p < 0.01 versus control. hpe: hours post-exposure.

Discussion

In this study, zebrafish were used to investigate isopsoralen hepatotoxicity. Isopsoralen affected the survival rate and hepatotoxicity of zebrafish larvae in a dose- and time-dependent manner. Moderate-dose (50 μM) and high-dose (100 μM) isopsoralen caused significant liver injury in the zebrafish larvae, which manifested as elevated liver transaminase (ALT and AST) levels, obvious liver atrophy, weakened liver fluorescence, unclear liver cell contours, vacuole formation and sparse cytoplasm. At the same time, after isopsoralen treatment, the zebrafish larvae showed decreased body length, missing fish swim bladder and delayed yolk sac absorption. The zebrafish yolk sac contains 70% neutral lipids, which are mainly metabolized in the liver, so the size of yolk sac can be used as an indicator of liver function.¹¹ In this experiment, the delayed yolk sac absorption was also a manifestation of the toxicity of isopsoralen that caused liver injury in the zebrafish larvae.

Based on the determination of isopsoralen hepatotoxicity in zebrafish larvae, the mechanism of this hepatotoxicity was investigated in this study. The results showed that isopsoralen could cause significant changes in the transcriptional levels of genes related to antioxidant capacity and drug transport function in the zebrafish larvae.

L-FABP, a long-chain and branched-chain fatty acid transport carrier in hepatocytes, is specifically distributed in the liver and is responsible for the transport of fatty acids as an important endogenous antioxidant during oxidative stress responses.^12,13 L-FABP can be used as a biomarker to monitor early hepatocyte damage.¹⁴ Glutathione S-transferases (GSTs) and superoxide dismutase (SOD) are very important antioxidant enzymes and oxygen free radical scavengers in the body that have spontaneous resistance to oxidative stress and can aid in the response to exotoxin- and reactive oxygen species-mediated toxicity and damage.^15,16 In these experiments, it was found that isopsoralen could inhibit the expression of lfabp, GST encoding gene gstp2 and SOD encoding gene sod1 in a dose-dependent manner, suggesting that the molecular mechanism of isopsoralen-induced liver injury in zebrafish may be related to the decreased detoxification capacity of the antioxidant system, leading to oxidative damage in the body.

Previous studies have shown that some drugs or diseases can cause the changes in drug transporter expression and activity^17,18 and that the abnormal expression of the transporters is also associated with the occurrence of drug hepatotoxicity.^19,20 For example, Qu et al. investigated the effects of Diosbulbin B on the transporters Ntcp, Oatp2, Bsep and MRP2 in mice and found that the expression of MRP2 may play an important role in Diosbulbin B-induced liver injury.²¹ A study by Saab et al. showed that inhibition of hepatobiliary transporter activity (such as the important efflux transporters MDR1 and MRP2) plays an important role in drug-specific liver injury.²² Therefore, studying the effects of isopsoralen on drug transporters is important to understand the mechanism of isopsoralen-mediated hepatotoxicity. The results of this study showed that the mRNA expression levels of mdr1, mrp1 and mrp2 in the tissue of zebrafish larvae were significantly down-regulated by isopsoralen, indicating that treatment with isopsoralen could significantly inhibit the expression of drug transporters in the zebrafish larvae, thus reducing the efflux effect of isopsoralen from the hepatocytes, leading to intracellular drug accumulation and hepatocyte injury.

In conclusion, this study investigated for the first time the hepatotoxicity of isopsoralen in liver fluorescent transgenic zebrafish. Isopsoralen was hepatotoxic to zebrafish larvae possibly due to isopsoralen inhibition of drug transporter expression, resulting in isopsoralen accumulation in the body and decreased antioxidant capacity, leading to liver injury. The study of changes in drug transporters provided a new direction for the study of hepatotoxicity of isopsoralen. The molecular mechanism of ispsoralen on zebrafish transporters needs further study.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (grant nos. 81703624 and 81703790), Natural Science Foundation of Shandong Province (grant nos. ZR2015YL010 and ZR2016YL009) and National Key Research and Development Project (grant no. 2016YFE0111600).

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