Effect of Pachybasin on General Toxicity and Developmental

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Effect of pachybasin on general toxicity and developmental toxicity in vivo Yi-Ruu Lin, Ming-Huan Chan, Huan-Lin Peng, Kou-Cheng Peng, and Shu-Ying Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03879 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Journal of Agricultural and Food Chemistry

Effect of pachybasin on general toxicity and developmental toxicity in vivo Yi-Ruu Lin1, Ming-Huan Chan2, Huan-Lin Peng3, Kou-Cheng Peng1*, Shu-Ying Liu3* 1

Department of Life Science and Institute of Biotechnology, National Dong Hwa

University, Hualien 97401, Taiwan 2

3

Institute of Neuroscience, National Chengchi University, Taipei, 11605, Taiwan Department of Molecular Biotechnology, Da-Yeh University, Changhua 51591, Taiwan

*Co-Corresponding author: Kou-Cheng Peng (Tel: +886-3-8633635, Fax: +886-3-8633630, E-mail: [email protected]) *Corresponding author: Shu-Ying Liu (Tel: +886-4-8511888 ext4256, Fax: +886-4-8511326, E-mail: [email protected])

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ACS Paragon Plus Environment


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ABSTRACT

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In order to document the safety of pachybasin, a secondary metabolite of Trichoderma

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harzianum, for use as a bio-agricultural agent, it was subjected to general toxicological

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testing in mice and developmental toxicity in zebrafish. With either 5 or 20 mg kg-1

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pachybasin i.p. injection, mice behavioral responses such as motor coordination,

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spontaneous locomotor activity, or nociceptive pain was not influenced. In long-term effect

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(daily injection for 14 days), the physiological, haematological, liver, and kidney functions

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were not altered either. Evidence for the developmental toxicity of pachybasin (10-100 µM)

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in 72-h exposure period was shown in zebrafish larvae, based on developmental retardation,

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impairment of chorion and increase of mortality. In summary, there are no significant

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general toxicities presented in the pachybasin-treated adult male mice. However, the

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embryo-toxicity in aquatic biota should be taken into consideration during bio-agricultural

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agent application.

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Keywords

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Pachybasin, Trichoderma harzianum, general toxicity, developmental toxicity.

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INTRODUCTION

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Trichoderma spp. is the important agricultural biological control agent against

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phytopathogenic fungi such as Rhizoctonia solani and Botrytis cinerea. The secondary

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metabolites of Trichoderma conferred its biocontrol activity either directly by inhibiting

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pathogens (so-called direct antagonism) of the host plant, or indirectly by inducing host

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plant resistance1, 2. Pachybasin, 1-hydroxy-3-methyl-anthraquinone (Figure 1), had been

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isolated and purified with high yield in T. harzianum Th-R16 and T. harzianum ETS

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323.1, 3 This compound had displayed antifungal activity against phytopathogenic fungi,

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such as R. solani and B. cinerea.1 It also played a role of self-regulation in T. harzianum

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mycoparasitic coiling.4 These characteristics make pachybasin a potential candidate for

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field application. Thus, it is important to evaluate the environmental safety of pachybasin.

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In the rodent brain experiments, emodin and emodin-8-O-beta-D-glucoside, derivatives

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of anthraquinones, have been shown to exhibit neuro-protective effects against ischemic

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brain damage in in vitro and in vivo models apparently by indirectly attenuating glutamate

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release or directly inhibited the neuronal damage induced by glutamate.5, 6 Another aspect,

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emodin had been proved may afford a significant neuroprotective effect against

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glutamate-induced apoptosis through activation of the PI3K/Akt signaling pathway, and

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subsequently enhance behavioral function in cerebral ischemia. 7 It has been reported that

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aleo-emodin could inhibit NMDA-induced apoptosis in retinal ganglion cell.8 However,

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emodin did not change the glutamate receptor-induced potential.5 Glutamate receptors are

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important for normal central nervous system (CNS) functions so that glutamate antagonists

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possess the serious adverse effects, such as motor incoordination, memory impairment and

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psychotomimetic effects.9 Furthermore, emodin (> 6,000 ppm) was demonstrated to induce

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mice developmental toxicity during gestation day (GD) 6-17.10 Emodin administered alone

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also did not influence on horizontal or vertical locomotor activity in rats.11, 12

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Pachybasin may possess a favorable ability to be a bio-agricultural agent. However, 3



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there is no published study on the safety evaluation of this chemical. In the present study,

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we evaluated the general toxicity and limiting glutamate excitotoxicity of pachybasin in

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mice, its embryo- and larvae-toxicity in zebrafish was characterized as well. In whole

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animal tests, acute exposure to pachybasin had no effect upon motor coordination,

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nociceptive threshold and locomotor activity of mice, and subacute exposure had no effect

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on growth rates, organ weight, and liver and kidney functions in mice, however, slight

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diarrhea was observed. Furthermore, it induced development toxicity and conformation

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impairment of chorion in embryos of zebrafish.

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MATERIALS AND METHODS

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Chemicals. Pachybasin was isolated from T. harzianum Th-R16, as previously described.1

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Potassium chloride (KCl), sodium chloride (NaCl) and magnesium sulfate (MgSO4) were

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purchased from J.T. Baker (Mallinckrodt Baker, Inc, Kentucky, USA). Other chemicals

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were obtained from Sigma (St. Louis, MO).

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Toxicity Test in Rodents. Animal. The male NMRI mice (8–9 weeks, 31–43 g) were

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supplied from the Laboratory Animal Center of Tzu Chi University (Hualien, Taiwan) and

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housed 4–5 animals per cage in a 12 h light/dark cycle with free access to food and water.13

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All experimental procedures followed the Republic of China Animal Protection Law

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(Chapter III: Scientific Application of Animal) and approved by Review Committee of the

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Tzu Chi University. The control group received an i.p. injection of 10 mL corn oil per kg

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while the pachybasin groups received 5 or 20 mg pachybasin dissolved in corn oil per kg.

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Since the water solubility of pachybasin is very low, 0.00019 g/l at 20 °C (SDS,

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Sigma-Aldrich), 20 mg/kg is the maximum testing concentration allowed for preparation.

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Acute Toxicity Test. The effects of the pachybasin on motor coordination were

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determined by the inverted screen test and the chimney test.14 For the inverted screen test,

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each mouse placed alone on the top of 2.0 cm2 wire mesh screen elevated 30 cm above the

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tabletop. The screen slowly rotated by 180o so that the mouse was suspended upside down

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on the bottom of the screens. The following behavioral responses were exhibited scores

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over a 60 sec testing session: climbed to top = 0; failed to reach top but held onto screen =

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1; fell off from the screen = 2. For the chimney test, the mice were inserted in a rugged

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tube (3 cm i.d., 25 cm long). Once the mice reached the opposite end, the tube was rapidly

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changed from horizontal to vertical and the timer was started. The timer was stopped after

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the mice completely left the tube. The withdraw latencies of the chimney test or scores of

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the inverted screen test were measured before and after (15, 30 and 60 min) i.p. injection of

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the vehicle (10 mL kg-1) or the pachybasin (5 or 20 mg kg-1). 5



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The effects of pachybasin on physiological pain were determined by the tail-flick and

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the hot-plate paw-shaking tests as previously described.13, 15 Five or 20 mg kg-1 of

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pachybasin was i.p. injected. All experiments were conducted in duplicates.

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NMRI mice were placed into a darkened activity cage (Columbus Auto-Track System,

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Version 3.0 A, Columbus Institute, Columbus, OH, USA) for 60 min habituation. Then i.p.

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injected with pachybasin (5 or 20 mg kg-1) or the vehicle (10 mL kg-1) and monitored the

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distance (centimeter) traveled for 60 min. A 5% alcohol solution was used to clean the

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inner surface of the apparatus between trials to remove any potentially interfering odors

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left by the previous mouse.

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Subacute Toxicity Test. The mice were i.p. administered of pachybasin (5 or 20 mg kg-1)

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or vehicle (10 mL kg-1) daily for 14 days.12 Body weight and the general health were

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monitored daily. At the end of the 14 day-dose schedules, the blood samples were collected

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by capillary from eyehole. Serum biochemical parameters including glutamic-pyruvic

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transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), and urea nitrogen were

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assayed using the Roche Hitachi 717 Chemistry Analyzer (Nissei Sangyo Co. Ltd., Japan.).

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After CO2 inhalation sacrifice, brain, heart, lung, kidney, liver and spleen were weighted

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and compared between groups.

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Toxicity Test in Zebrafish. Zebrafish Maintenance and Eggs Collection. Zebrafish (AB

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strain, from TAIKONG corporation, Taipei, Taiwan) kept at the VIVO laboratory facility

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at least 3 weeks prior to the first intended spawning. The fish were raised and maintained

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on a 14:10 h light-dark cycle at 22-26 °C. The water was continuous aerated and renewal

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of the water occurred in a semi-static manner. Commercial dry flake food, frozen

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crustaceans or midge larvae were given as food twice per day.

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Toxicity Assay and Microscopic Observation. For embryo-larvae toxicity assay, the 30

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fertilized eggs of 2 hpf stages were randomly distributed into each well filled with 2 mL E3

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embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) of 6 well 6


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plate. Three concentrations (1, 10, and 100 µM) of pachybasin and a vehicle (1% DMSO)

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were set and each exposure solution was half renewed daily (due to the low water

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solubility of pachybasin, 100 µM is the maximum testing concentration allowed for

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preparation). Each plate was covered and incubated at 28±0.5 °C throughout the 72 h

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exposure period. The embryos were observed every 24 h through day 3 (72 hpf) and the

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occurrence of embryo-toxicity and morphological characteristics of the embryos was

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recorded and using an inverted light microscope (Eclipse TE-300; Nikon, Japan). The

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embryos were evaluated for the presence and morphological development of somite, tail

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detachment and chorion. After hatching, the kinks in the tail and sidewise position were

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observed.

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For larvae toxicity assay, 3 larvae of 5 dpf stages were randomly distributed into each

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well filled with 2 ml E3 embryo medium of 6-well plate. Three concentrations (1, 10 or

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100 µM) of pachybasin and a vehicle (1% DMSO) were set and the exposure solution

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was half renewed daily. The plate was covered and incubated at 28±0.5 °C. The larvae

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were evaluated for the mortality throughout the 7-day exposure period.

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Statistical Analysis. All data were expressed as means ± SEM. Statistical significance of

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difference between groups was determined by the two-way repeated measures ANOVA or

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the one-way ANOVA followed by the Student-Newman-Keuls post-hoc test. A P value of

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less than 0.05 was considered statistically significant.

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RESULTS

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Pachybasin Had no Acute Toxicity in NMRI Mice. Motor Coordination. Pachybasin (5

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and 20 mg kg-1, i.p.), given 60 min prior to the experiments, did not cause any significant

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changes both in scores of the inverted screen [F(2, 99)=1.373, P=0.274] and the latency

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response of chimney test [F(2, 99)=2.298, P=0.124] (Table 1).

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Nociceptive Effects. Pachybasin (5 and 20 mg kg-1, i.p.) had no significant effects on

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the hot-plate paw-shaking assay [F(2, 83)=0.627, P=0.545] at 15, 30, 60, and 120 min after

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testing agent application. Similarly, pachybasin (5 and 20 mg kg-1, i.p.) did not alter the

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withdrawal latency for tail-flick [(F2,94) = 0.0387, P = 0.962] (Table 2).

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Locomotor Activity. No significant difference was found on the total distance traveled

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by the mice treated with pachybasin (5 and 20 mg kg-1, i.p.) compared to the vehicle group

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[F(2, 21) = 0.998, P = 0.387] (Figure 2).

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Pachybasin Had no subacute Toxicity in NMRI Mice. Body Weight Trends and Clinical

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Character Observation. Figure 3 showed that the body weight gain of mice received daily

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injection of pachybasin (5 and 20 mg kg-1, i.p.) for 14 days duration was not different from

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the vehicle group. Although, 75% of mice exhibited chronic diarrhea in response to 20 mg

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kg-1 pachybasin, however, this phenomenon disappeared completely in the 5th dose

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treatment. In general, the mice remained in good health throughout the subacute study.

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Subacute Administration of Pachybasin Had No Adverse Effect on Organ Weights.

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The wet weights of mouse organs such as brain, heart, lung, liver, kidney and spleen

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showed no significant differences between the control mice and those i.p. administrated

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with pachybasin at 5 and 20 mg kg-1 for 14 consecutive days (Table 3).

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Effects of subacute Administration of Pachybasin on Liver and Renal Function. The

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influence of pachybasin to liver and kidney functions of mice were determined

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biochemically in serum GOT, GPT, BUN and creatinine activities. There were no

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significant differences between the i.p. administration of the pachybasin at 5 and 20 mg 8


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kg-1 for 14 consecutive days vs. the i.p. vehicle injected control mice (Table 4).

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Pachybasin-Induced Developmental Toxicity in Zebrafish Embryos. Two hours after

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fertilization, the embryos were exposed to 1% DMSO or pachybasin at 1, 10, and 100

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µM for 74h. Physical malformations were studied at specified stages (Figure 4A). From

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26 hpf, the developmental abnormalities were observed at 1, 10 and 100 µM

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pachybasin-treated embryos, mainly developmental retardation. From 50 hpf, zebrafish

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blastula with chorion (outer egg membrane) partially removed or same embryo

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completely removed from chorion at 100 µM pachybasin-treated embryos. Survival rates

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of 1, 10 and 100 µM pachybasin-treated embryos at 98 hpf after treatment decreased in a

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dose-dependent manner (Figure 4B). Results also showed the pachybasin-increased

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notochord distortions in embryos surviving in a dose-dependent manner (Figure 4C).

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Pachybasin Had No Lethal Toxicity on Zebrafish Larvae. The zebrafish larvae (5 pdf)

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exposed to 1% DMSO and pachybasin at 1, 10, and 100 µM showed no significantly

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lethal toxicity under consecutive 7-day treatment (Table 5).

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DISCUSSION

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Agricultural agents have contributed in increasing the production of crops, but their

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inappropriate use has caused serious problems like environmental pollutions and toxicity

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to living organisms. Biocontrol agents have the characteristic of target specific,

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environmental friendly and biodegradability. Pachybasin is one of the anthraquinones

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obtained from a promising biocontrol agent of T. harzianum Th-R16.1 Anthraquinones

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possess remarkable laxative property. The laxative effect of anthraquinones is caused by

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the changing of colonic motility and alteration in colonic absorption and secretion.15

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Emodin exhibited stimulatory actions on gastrointestinal smooth muscle have been

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described in several studies. 7, 16 It is believed that the presence of hydroxyl groups at

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positions 1 and 8 on the aromatic ring of emodin are essential for laxative action.17 In this

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study, 75% of mice exhibited chronic diarrhea in response to 20 mg kg-1 pachybasin,

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however, this phenomenon disappeared completely in the 5th dose treatment. We

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speculated that the attenuated laxative properties of pachybasin are mediated by the

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presence of a hydroxyl group at position 1 on the aromatic ring as well as emodin.

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Previous studies have demonstrated that anthraquinones, such as aleo-emodin could

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inhibit NMDA-induced apoptosis in retinal ganglion cell8. However, emodin did not

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change the glutamate receptor-induced potential.5 On the other hand, emodin and

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emodin-8-O-beta-D-glucoside possess the ability to inhibit tonic release of glutamate in

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vitro and in vivo, limit glutamate excitotoxicity in animal models of brain damage.5, 6 In

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mammals, innumerable behaviors including gross motor coordination, locomotion and

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exploration are regulated, in part, by the physiological regulation of glutamate, the

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principal excitatory neurotransmitter in the mammalian brain.9 Convincing evidence has

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demonstrated that the development of central hyperexcitability and persistent pain are

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associated with the activation of glutamate receptors.18 Glutamate receptor antagonists,

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such as MK-801, ketamine or AP5, intrathecally applied can attenuate the nociceptive 10


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behaviors in several animal models.19, 20 However, emodin has been administered that

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alone also did not influence on horizontal or vertical locomotor activity in rats. 11, 12 We

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observed that acute and subacute administration of pachybasin induced no behavioral

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abnormalities in motor coordination, nociceptive threshold and locomotor activity of mice.

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In the 1960’s, a study indicated that the chronic abuse of anthraquinones in rhubarb

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extract was associated with an increased incidence of liver cirrhosis and hypokalemia.21

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However, several evidences showed the positive potential effects of emodin and

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emodin-8-O-beta-D-glucoside on gastrointestinal and renal systems.22, 23 Recent studies

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in mice infected with prostate cancer or neuroectodermal tumors demonstrated that

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emodin- and aloe-emodin-treated mice had no appreciable acute and chronic toxicity and

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exhibited significant longer survival and better body weight gain than the control group.24,

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Our investigations demonstrated that 14 days administration of pachybasin to healthy

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mice did not compromise weight gain, serological indicators of hepatic (GOT and GPT)

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and renal (creatinine and BUN) function or wet organ weights. Collectively, these results

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suggest that subacute administration of pachybasin in tested dose had no adverse effects

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upon behavior, gastrointestinal, hormonal and central or autonomic nervous systems in

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healthy mice.

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In animal, body weight change is a simple yet sensitive indicator for the adverse effects.

Embryogenesis is the most sensitive stage to toxicant exposure; chemicals registered

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for commercial use should possess the developmental safety data. Recent studies evaluated

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the developmental toxicity of emodin in rats and mice, the results demonstrated the lowest

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observed adverse effect levels were 1,700 ppm and 6,000 ppm, respectively, based on

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reduction of fetal body weight.10, 26 The zebrafish system has become one of the models of

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selection for research on developmental toxicity because of chemical permeability, optical

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transparency of the zebrafish embryo, physiological similarity to mammals and sensitive to

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environmental changes. In recently studies, zebrafish embryo had been generally used to 11



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appraise the teratogenic toxicity of variform agricultural agents.27-29 Our investigations

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demonstrate that pachybasin induced developmental toxicity in embryo stage, based on

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developmental retardation, impairment of chorion, notochord distortions and increase of

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mortality. Although the mechanism of pachybasin-induced developmental toxicity was still

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unclear, but the developmental retardation may permit teratogens to act for a longer time

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during sensitive stages and thus intensify the severity of the malformations and mortality

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produced. The notochord is an axial structure common to the phylum of the chordates. It is

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a primary axial structure for proper differentiation of adjoining tissues such as

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neurectoderm, muscle and vertebral elemental. Our investigations showed that pachybasin

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induced notochord distortions in zebrafish embryos. Previous studies have demonstrated

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that various agricultural agents-induced disrupt normal morphogenesis and differentiation

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of the notochord may therefore result in permanent skeletal deformities, muscle

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abnormalities, overactive muscle spasms and neurological dysfunction.28-30 The chorion of

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zebrafish provides a protective barrier between the developing embryo and many chemical

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pollutants, such as agricultural agents. Although the mechanisms of pachybasin-induced

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impairments of chorion were still unclear, the impairment of chorion may lead to the

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developmental toxicity and mortality in zebrafish. However, pachybasin did not induced

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lethal toxicity in zebrafish larvae. These results indicate that pachybasin is potent

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teratogens during early zebrafish development.

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In conclusion, this study presents the first safety assessment of pachybasin conducted

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on mice and zebrafish. It showed that pachybasin had no effect upon motor coordination

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and locomotor activity of mice. Subacute exposure of pachybasin had no effect on growth

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rates in mice, although slight diarrhea was observed. However, the embryo-toxicity in

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aquatic biota should be taken into consideration during bio-agricultural agent application.

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Abbreviations

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NMDM: N-methyl-D-aspartate; i.p.: intraperitoneal injection; % MPE: percentage of 12


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maximum possible effect; GPT: glutamic-pyruvic transaminase; GOT:

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glutamic-oxaloacetatic transaminase; hpf: hours post fertilization; dpf: days post

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fertilization; BUN blood urea nitrogen; DMSO: dimethyl sulfoxide.

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Competing interests

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The authors declare no competing financial interest.

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Authors' contributions

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YRL carried out general toxicity, and drafted the manuscript. MHC

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coordinated the experiments and manuscript modification. HLP provided the

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technical and material support. SYL and KCP conceived the study scheme and helped to

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draft the manuscript. All authors read and approved the final manuscript.

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Funding

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This work was supported by grants MOST 104-2311-B-259-001 and MOST

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105-2313-B-212-001 from the Ministry of Science and Technology, Taiwan, ROC.

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FIGURE CAPTIONS

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Figure 1. Structure of pachybasin.

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Figure 2. Effects of pachybasin on locomotor activity. Mice were habituated in testing

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cages for 60 min and challenged with vehicle or pachybasin (5 and 20 mg kg-1, i.p.).

345

The total distances traveled were recorded for 60 min. Mean ± SEM (n=8). Data

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were analyzed by one-way ANOVA followed by Student–Newman–Keuls post-hoc

347

test.

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Figure 3. Body weight curves of vehicle- and pachybasin-treated mice. Values are means

349

± SEM (n=8-9). Data were analyzed by two-way repeated measures ANOVA

350

followed by Student–Newman–Keuls post-hoc test.

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Figure 4. Development toxicity of embryos following pachybasin exposure from 2 to 74

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hpf. (A) Embryo exposure to vehicle or pachybasin (1, 10 or 100 µM) exhibited

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morphological abnormalities in 26, 50 and 74 hpf. (B) Pachybasin significantly

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reduced survival rate in embryos. (C) Pachybasin significantly increased notochord

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distortions rate in embryos. Mean ± SEM (n=4-5). Data were analyzed by one-way

356

ANOVA followed by Student–Newman–Keuls post-hoc test.

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Table 1. Effects of Pachybasin on Inverted Screen and Chimney Test in Miceab

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Table 2. Effects of Pachybasin on Hot-Plate and Tail-Flick Test in Miceab

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Table 3. Effects of Pachybasin on Organ Weight in Micea

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Table 4. Effects of Pachybasin on Hepatotoxicity and Renal Toxicity in Micea

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Table 5. Effects of Pachybasin on Alevin Toxicity in Zebrafisha

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TOC graphic

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Effect of Pachybasin on General Toxicity and Developmental

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