Hematopoiesis toxicity induced by 4-methylumbelliferon determined in an invertebrate model organism.

Abstract Umbelliferone has potential value as it has an inhibitory effect on tumor cells; however, its impact on an animal's circulatory system and hematopoietic function has not been reported. In this study, 4-methylumbelliferon (4-MU), an umbelliferone derivative, was used as a model drug, and its potential toxicity on hemocytes and hematopoietic organs (HOs) was investigated using an invertebrate animal model, the silkworm, Bombyx mori. The results showed that the level of reactive oxygen species in HOs increased when larvae (third day of the fifth instar) were orally exposed to 4 mM 4-MU for 8 min, followed by the induction of improved antioxidative metabolism of coenzymes in hemolymph. Exposure to 4-MU also significantly upregulated the expression levels of several genes in the hemolymph and fat body (a detoxification tissue similar to the liver in mammals) including antimicrobial peptide gene cecropinA and moricin, and a phagocytosis-related gene, tetraspanin E, suggesting an increased antioxidant level and antimicrobial ability of the circulatory system. However, the percentage of dead hemocytes increased and hematopoiesis significantly decreased in HOs, indicating the toxic effect of 4-MU on hemocytes and hematopoiesis, despite it inducing enhanced antioxidant and antimicrobial activity in the circulatory system.

Introduction 4-methylumbelliferon (4-MU) is an umbelliferone derivative and is used as a model drug to study the pharmocodynamics of umbelliferones. 4-MU is rapidly metabolized into harmless substances by glycosylation and sulfation in mice, but produces different metabolites in different tissues (Chen & Pang, 1997). In the invertebrate model, Bombyx mori (the silkworm), 4-MU is mainly glycosylated (Hamamoto et al., 2009), which may be related to the extremely strong activity of glycosylase in the digestive tract and hemolymph of silkworms (Xu, 2013). In recent years, a large number of in vitro studies using mouse and human cells have reported the antitumor effect of 4-MU (Bhattacharyya et al., 2009;Oemardien et al., 2011;Twarock et al., 2011;Urakawa et al., 2012). One possible mechanism is that 4-MU may inhibit the synthesis of hyaluronic acid, an important marker for tumor development (Nakazawa et al., 2006). The double bonds which exist between carbon atoms and oxygen atoms in derivatives of the coumarin family produce reactive oxygen species (ROS) in vivo (Kuki et al., 2008). We previously observed that oral exposure to 4-MU induced a considerable increase in ROS (mainly H 2 O 2 ) in gastrointestinal cells, increased antioxidant levels and the antibacterial ability of the digestive tract in B. mori, protected the tissues from injury (Fang et al., 2014a), and prevented cell damage (Fang et al., 2014b) in the digestive tract, indicating a new in vivo pathway for 4-MU.
Hemocytes are a suitable model for the study of innate immune response and DNA damage in vivo (Irving et al., 2005;Cherry & Silverman, 2006;Carmona et al., 2011). However, there have been no reports on the effect of umbelliferones on hematopoietic function. Bombyx mori is an ideal animal species for in vivo and in vitro research on the effects of toxins on hematopoiesis. The generation and release of hemocytes in hematopoietic organs (HOs) can be reliably repeated in vitro (Liu et al., 2014). Nearly all organs and tissues in insects such as flesh fly (Kurata et al., 1989) and B. mori (Liu et al., 2014) are immersed in hemolymph, and the results from in vitro cell culture experiments are close to the results from in vivo animal experiments. Thus, the limitations arising from tissue barriers in mammals can be avoided (Tsoi et al., 2013). This study assessed how 4-MU differentially acted on hemocytes and generated cytotoxicity.
In particular, we determined whether 4-MU had a positive impact on the development of hematopoietic tissue and hematopoiesis function.

Preparation of animals
The Dazao strain of B. mori was reared at 25 C with a photoperiod of 12 h light and 12 h dark. Similar-sized larvae (body weight 1.0 ± 0.1 g) were orally exposed to 4-MU (Sigma, St. Louis, MO, USA) on day 3 of the fifth instar. Following the method of Fang et al. (2014b), 4-MU was dissolved in 10% dimethyl sulfoxide (DMSO) to a final concentration of 4 mM. The instillation dose was 50 mL per individual. In order to minimize food residue, the animals were starved for 3 h before exposure to 4-MU; animals in the control group were also starved.

Assessment of hemocytes and hematopoiesis
Hemolymph was collected after the pereiopods were punctured using needles and placed in a centrifuge tube at 8, 30, 60, 120 and 180 min after 4-MU injection. A volume of 100 mL hemolymph was used for hemocyte acridine orangepropidium iodide (AO/PI) staining, and the remainder was used for biochemical assays and gene expression profile assays.
Following the method of Liu et al. (2014), the HOs were carefully removed using surgical forceps. The HOs were washed with precooled physiological saline and Grace's insect tissue culture medium. A hanging drop culture was performed for each HO. The culture medium used was Grace's insect tissue culture medium supplemented with 10% B. mori hemolymph and an appropriate amount of antibiotics. The volume of the culture medium was 10 mL, and the culture temperature was 25 C.

AO/PI staining
The hemocytes were double stained with AO/PI in order to assess cell apoptosis. AO (Item No. A742007) and PI (Item No. PB1112) were purchased from Sangon Biotech Co., Ltd (Shanghai, China).

Analysis of gene expression profiles
Total RNA was isolated from five larvae hemolymph in each group using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and purified using an Oligotex Õ mRNA Midi kit (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. The real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to investigate the expression level of genes using the primers listed in Supplementary Table S1. An SYBR Õ Premix Ex TaqÔ kit (Perfect Real Time) was used for qRT-PCR and Ribosomal protein49 (Rp49) and Elongation2 (Elo2) of B. mori was set as the reference genes. The reaction program was as follows: 95 C degeneration for 1 min, followed by 40 cycles at 95 C for 5 s, 58 C for 10 s, and 72 C for 10 s. The process was set automatically by the detection program and three replicates were used for each.

Superoxide anion and reactive oxygen species (ROS) staining
The levels of superoxide anion and ROS were measured using a dihydroethidium (DHE) kit (Beyotime, Shanghai, China) and ROS kit (Beyotime, Shanghai, China), respectively. The HOs was collected in diethypyrocarbonate (DEPC) (containing 0.7% NaCl) to avoid air exposure and was then vortexed for 20 s in normal saline which included 0.1% DMSO. The HOs were then quickly placed into a staining solution for 7 min (for DHE) or 15 min (for 2 0 ,7 0 -dichlorofluorescin diacetate, DCFH-DA), and then washed with saline for 5 min in the dark room. The red fluorescence (for superoxide anions) or green fluorescence (for ROS) was observed using a fluorescence microscope (Olympus Medical Systems Corp., Tokyo, Japan).

Replication of experiments and statistical analyses
All experiments were performed at least three times, and are presented throughout as mean ± SD. The significant differences between control group and treatment group were determined using the Student's t-test. Data from multiple time points were analyzed by one-way ANOVA in combination with Origin7.5 Software.

4-MU exposure increased ROS levels in HOs and hemolymph
We previously demonstrated that 4-MU passed through the membrane barrier of the digestive tract and entered the circulatory system of silkworms 8 min after oral exposure to 4-MU (Fang et al., 2014a). In this study, HOs were collected 8-60 min after oral 4-MU exposure and subjected to ROS and superoxide anion staining. The results showed that the level of ROS in HOs increased 8-30 min after 4-MU injection ( Figure 1A) and the level of superoxide anion, a specific ROS, significantly increased 30 min after exposure ( Figure 1B), suggesting that 4-MU induced ROS other than superoxide anion in HOs.
It was impossible to collect a sufficient amount of hydrogen peroxide (H 2 O 2 ) for accurate analysis due to the small size of HOs. Furthermore, the hemolymph of silkworms was easily oxidized, which made it even harder to accurately measure H 2 O 2 levels. Therefore, we examined the activities of NADH peroxidase (NOX-1), a key enzyme in the synthesis of H 2 O 2 in hemolymph. As shown in Figure 1(C), NOX-1 activity was increased by 93% in hemolymph 16 min after 4-MU exposure, indicating that the level of H 2 O 2 , another important ROS, may have increased.

4-MU-induced metabolic changes in coenzymes in the hemolymph
As the activity of NOX-1 is related to coenzyme I and the metabolism of H 2 O 2 is indirectly affected by coenzyme II, the substrates and products associated with the coenzyme I and II system were measured (Figure 2). The results showed that the NADH/NAD + ratio increased by 3.56-3.78 times within 32 min after 4-MU injection (Figure 2A), returned to the control level after 48 min, and further declined to 21% of the control level at 120 min. The NADPH/NADP + ratio increased threefold with a lower frequency and smaller amplitude within 120 min after 4-MU exposure, and then quickly decreased to the control level ( Figure 2D). It is worth noting that NADPH and NADP + levels in the early stage (within 16 min) after 4-MU exposure were significantly lower than those in the control group, returned to the control levels at 32 min, and increased to much higher levels at 60 and 120 min compared with the control group ( Figure 2E and F).
The NADH/NAD + ratio is an indicator of the level of glycolysis and metabolism in the tricarboxylic acid (TCA) cycle. A higher ratio indicates that cells are in an overoxidized state with a higher level of oxygen consumption (Houtkooper et al., 2010). As shown in Figure 2(A, B and C), 4-MU-induced ROS quickly resulted in the hemolymph being in the over-oxidized state; however, the NADH/NAD + ratio was restored 48 min after exposure. As a reducing agent for  Figure 2(A, B and C) shows the mean ratio of NADH/ NAD + , contents of NADH and NAD + , respectively. One unit was defined as nmol of NAD + or NADH per milligram of tissue. Figure 2(D, E and F) shows the mean ratio of NADPH/NADP + , contents of NADPH and NADP + , respectively. One unit is defined as nmol of NADPH or NADP + per milligram of protein. Treatment was repeated 3 times, the animal number (sample size) of each hemolymph sample is 5 larvae, and each bar chart ± standard deviation represents mean value in triplicates. *p50.05 and **p50.01 indicate significant differences. , and (C) the activity of NADH peroxidase in hemolymph. Treatment was repeated 3 times, the animal number (sample size) of each hemolymph sample is 5 larvae, and each bar chart ± standard deviation represents mean value in triplicates. *p50.05 and **p50.01 indicate significant differences. DOI: 10.3109/01480545.2015.1079915 biosynthesis, NADPH is involved in the activation of enzymes such as catalase and glutathione peroxidase in the body, and indirectly affects the metabolism of H 2 O 2 . Therefore, the NADPH/NADP + ratio is commonly used to evaluate the redox state in vivo (Jeon et al., 2012). Figure 2(D, E and F) shows that NADPH was rapidly used against 4-MU-induced ROS in hemolymph, and thus a large amount of NADPH was consumed. Oscillation of the NADPH/NADP + ratio also suggested that NADPH oscillated due to its synthesis and consumption in hemolymph.

4-MU-induced apoptosis of hemocytes and hematopoiesis in HOs
As shown in Figures 1 and 2, exposure to 4-MU increased ROS levels in hemolymph and tissues in HOs, suggesting that oxidative damage in hemocytes and HOs may have occurred. Therefore, AO/PI staining of hemocytes was performed and the results demonstrated that the percentage of dead hemocytes significantly increased 30-60 min after 4-MU exposure ( Figure 3).
In order to clarify the reason for the increased percentage of dead hemocytes in hemolymph, we further investigated the expression levels of apoptosis-related genes in hemolymph. It was found that the mRNA levels of major genes involved in the apoptosis signaling pathway including, MAPK-ERK kinase (Mek) and extracellular regulated MAP kinase (Erk), slightly decreased within 3 h after 4-MU exposure. The mRNA level of p38 map kinase (p38) slightly increased for a short period of time followed by a slight decrease at 30 min, and a slight increase at 3 h after 4-MU injection ( Figure 3D). It has been reported that cell apoptosis occurs only when the expression of Mek and Erk simultaneously decrease and the expression of p38 is significantly upregulated in an organism (Iyoda et al., 2003). As shown in Figure 3(C), the increase in the percentage of dead cells in hemolymph after 4-MU exposure might not be entirely, or even mainly, induced by apoptosis of hemocytes. Alternative reasons may exist.
The hematopoietic capacity of HOs was further investigated after HOs were exposed in vivo to 4-MU and cultured in vitro (Figure 4). The results showed that although 4-MU-treated HOs exhibited no obvious atrophy and Live or dead hemocytes of prohemocytes, granulocytes and spherulocytes were stained green by AO and all hemocytes in a bright field. (D) Expression profile of apoptosis-related genes Mek, Erk and p38. In Figure 3(A, B and D), treatment was repeated 3 times, the animal number (sample size) of each hemolymph sample is 5 larvae, and each bar chart ± standard deviation represents mean value in triplicates. *p50.05 and **p50.01 indicate significant differences. X-axis shows the time after 4-MU exposure. apoptosis, these HOs had a much slower rate of hematopoiesis and produced significantly fewer hemocytes compared with the positive control (DMSO-treated HOs). HOs cultured in vitro beginning 30 min after 4-MU exposure had significantly lower hematopoietic capacity compared with those cultured earlier or later, indicating that the influence of 4-MU exposure on HOs continued to rise within 30 min, followed by self-repair of HOs. These observations were consistent with the changes in the percentage of dead hemocytes over time (Figure 3).

4-MU affected the expression of phagocytosis-related and antimicrobial peptide genes
It has been reported that umbelliferones have strong antibacterial effects on plant pathogenic bacteria in vitro (Zhang & Jiang, 2010). Our previous study showed that 4-MU can improve the antibacterial ability of the digestive tract once it enters the tract of B. mori (Fang et al., 2014b). The changes in the expression of antimicrobial peptide genes in the fat body ( Figure 5A and B) and hemolymph ( Figure 5C and D) were measured, and the results showed that both the hemolymph and fat body responded quickly to 4-MU exposure by upregulating the expression of antimicrobial peptide genes including cecropinA and moricin within 2 h after exposure. The expression levels of both genes were subsequently downregulated to normal levels. The expressed antimicrobial peptides (AMPs) in the fat body may be further secreted into hemolymph for their function. In other insect models (e.g. Drosophila), starvation is relative with immunity systemically (Brown et al., 2009). In this case, the animals were starved for 3 h before exposure to 4-MU. Compared to control group, the immune-related genes cecropin A and moricin were upexpressed in fat body and hemolymph within 3 h ( Figure 5), it is mainly induced by the 4-MU. AMPs have been shown to be highly toxic to bacteria and tumor cells with almost no toxicity in normal tissue cells (Shin et al., 2000;Sergio et al., 2003). The results shown in Figure 5 indicate that 4-MU had potential pharmacological effects in improving antibacterial and antitumor abilities by inducing the expression of AMPs in B. mori.
As 4-MU upregulated the expression level of antimicrobial peptide genes in hemolymph ( Figure 5), we speculated that phagocytosis of hemocytes may also be affected. The expression levels of phagocytosis-related genes were further examined. The results demonstrated that 4-MU exposure increased the expression level of tetraspanin E (TspE) gene without affecting the expression of Ced-6 (Ced-6) gene and ActinA1 (ActA1) gene (Figure 6), which suggested that 4-MU did not increase antibacterial ability by enhancing phagocytosis of hemocytes.

Discussion
Umbelliferones are widely found in plants, but have biological activities in many animal species and have been used for medicinal purposes (Morabito et al., 2010). These compounds also show strong antibacterial effects on plant pathogenic bacteria in vitro (Zhang & Jiang, 2010). In recent years, the  Figure 4(B). +++ indicates normal hematopoiesis, ++ indicates only a small amount of hematopoiesis and a further increase in abnormal hemocytes and + indicates very little or no hematopoiesis. Fifth-instar larvae were orally injected with 50 mL 4 mM 4-MU per individual for 48 h and control organisms were injected with the same volume of 10% DMSO. Exposure duration in vivo was the time to remove the HOs after exposure to 4-MU. Culture duration in vitro was the incubation time free from 4-MU. A single HO was cultured using the hanging drop method in 10 mL Grace's insect medium at 25 C with continuous illumination. ability of umbelliferones to inhibit the growth of cancer cells as well as the occurrence and development of tumors has been observed in many in vivo and in vitro experiments using mammalian cells including mouse and human cells (Bhattacharyya et al., 2009;Nakazawa et al., 2006;Oemardien et al., 2011;Twarock et al., 2011;Urakawa et al., 2012). Our previous research strongly suggested that 4-MU exposure induced a considerable increase in H 2 O 2 , and increased antioxidant levels and the antibacterial ability of digestive tract in B. mori (Fang et al., 2014b). Our experimental results also provide evidence to show that only trace amounts of 4-MU were observed in the fat body, a detoxification tissue similar to the liver in mammals, after exposure to 4-MU by oral injection, but strongly increased the ROS level and improved the antioxidant capacity in fat bodies, protected the tissues from injury and resulted in activation of the antioxidant enzyme system (Fang et al., 2014a). Similar antioxidant protection against cell damage was found in the digestive tract (Fang et al., 2014b).
In this study, oral 4-MU exposure induced changes in ROS levels and antioxidant capacity in hemolymph and HOs, which were similar to those observed in the digestive tract and fat body. It was also found that the expression levels of antimicrobial peptide gene, cecropinA and moricin, and phagocytosis-related gene, tetraspanin E, were significantly upregulated, indicating that antioxidant levels and the antibacterial ability of the circulatory system were simultaneously enhanced. However, the percentage of dead hemocytes in hemolymph significantly increased and hematopoietic capacity of HOs dramatically decreased within a short period of time.
Conclusion 4-MU has potential pharmacological effects in improving antibacterial and antitumor ability by inducing the expression of AMPs in B. mori. However, it has simultaneous potential toxicity on circulating hemocytes and their hematopoietic function.

Declaration of interest
The authors declare no conflict of interest.
This work was supported by the National Natural Science Foundation of China (Grant No. 31172264), National Natural Science Foundation of Jiangsu province (Grant No.