Semicarbazide-sensitive amine oxidase kills African trypanosomes in vitro

Acta Tropica 117 (2011) 161–164

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Semicarbazide-sensitive amine oxidase kills African trypanosomes in vitro Qiao-Ping Wang, De-Hua Lai, Zhi Li, Feng-Jun Li, Zhao-Rong Lun ∗ Center for Parasitic Organisms, State Key Laboratory of Biocontrol, School of Life Sciences, and Key Laboratory of Tropical Diseases Control of Ministry of Education, Zhongshan Medical College, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, PR China

a r t i c l e

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Article history: Received 22 June 2010 Received in revised form 25 November 2010 Accepted 25 November 2010 Available online 8 December 2010 Keywords: SSAO Trypanosome Formaldehyde Hydrogen peroxidase In vitro

a b s t r a c t The African trypanosome Trypanosoma brucei is the cause of sleeping sickness in humans and Nagana in animals. Here we report that semicarbazide-sensitive amine oxidases (SSAOs), enzymes that are abound in T. brucei mammal hosts, eliminate trypanosomes by oxidation of its substrate in vitro. SSAO and its endogenous substrate methylamine are not toxic to T. brucei, but parasites were killed in the presence of both of them. SSAO inhibitors antagonized the SSAO-methylamine induced toxicity on T. brucei. The trypanocidal activity was mainly associated with formaldehyde generated in the SSAO mediated oxidation of methylamine. This finding suggests that SSAO may play some roles in non-specific defense of trypanosome infection in mammals. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Protozoan parasites are susceptible to products generated during the oxidation of polyamines mediated by polyamine oxidase (PAO) and the oxidation of xanthine by xanthine oxidase (XO) (Ferrante et al., 1982,1984; Muranjan et al., 1997; Rzepczyk et al., 1984; Wang et al., 1999). In cattle trypanosomiasis, Trypanosoma brucei, is killed by the oxidation product of XO, hydrogen peroxide (H2 O2 ) (Muranjan et al., 1997; Wang et al., 1999) which plays an important role in control the parasitaemia in the early infection in African Cape buffalo (Wang et al., 1999). Beside XO and PAO, source of antiparasitic H2 O2 could also come from other oxidases. For instance, semicarbazide-sensitive amine oxidases (SSAOs) exist in many mammalian species, catalyzing primary amines into aldehyde, hydrogen peroxide and ammonia (O’Sullivan et al., 2004). The physiological substrates of SSAOs include methylamine, aminoacetone, tyramine, 2-phenylethylamine and 5-hydroxytryptamine (O’Sullivan et al., 2004; Yu et al., 2003). SSAOs present in both tissue-bound isoforms and soluble isoforms (plasma SSAO) (O’Sullivan et al., 2004). Soluble SSAOs originate from tissue-bound SSAOs (Stolen et al., 2004). Tissue-bound SSAOs are mostly located on the membrane of smooth muscle cells, adipocytes and vascular endothelial cells. Vascular adhesion protein-1 (VAP-1), an adhesion protein in endothelial cells, displays SSAO activity (Stolen et al., 2004).

∗ Corresponding author. Tel.: +86 20 84115079; fax: +86 20 84036215. E-mail address: [email protected] (Z.-R. Lun). 0001-706X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2010.11.010

SSAOs-mediated oxidative deamination is toxic to endothelial cells and vascular smooth muscle cells (Yu and Zuo, 1993, 1996; Hernandez et al., 2006). SSAOs are involved in the development of vascular damages in diabetes, heart failure, inflammation and even Alzheimer’s disease (AD) (O’Sullivan et al., 2004). Here we report that SSAO mediated oxidation of methylamine killed T. brucei in vitro. 2. Material and methods 2.1. Preparation of human umbilical artery SSAO and rVAP-1 Human umbilical SSAOs were isolated as previously described (Yu and Zuo, 1996). Briefly, umbilical arteries were washed three times with chilled phosphate buffer (0.01 M, pH 6.8) and sliced into small pieces, and then were homogenized by FS-2 Polytron homogenizer (Jintan, China) in the same chilled buffer. The crude homogenate was centrifuged at 800 × g for 10 min and the resulting supernatant was subjected to ultracentrifugation at 32 000 × g for 30 min. The final supernatant containing soluble SSAO was sterilized through a 0.22 ␮m filter and stored at −80 ◦ C. The recombinant human VAP-1 (rVAP-1) was a gift from Biotie Therapies, Turku, Finland. 2.2. Trypanocidal assays Bloodstream forms of T. brucei brucei strain STIB 920 were cultured in HMI-11 medium at 37 ◦ C with 5% CO2 for trypanocidal assays. To determine the antiparasitic effects of SSAO,

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Time(h) Fig. 1. rVAP-1 kills trypanosomes. Parasites were treated with methylamine, or rVAP-1 and methylamine. rVAP-1 and methylamine concentrations are 1.5 ng ␮l−1 and 1 mM, respectively. () Methylamine; and (♦) methylamine + rVAP-1.

parasites were incubation with SSAO/rVAP-1 (1.5 ng ␮l−1 ) or methylamine hydrochloride (1 mM) (Sigma) or both of them in the presence or absence of SSAO inhibitors (E)-2-(4-fluorophenethyl)3-fluoroallylamine hydrochloride (MDL-72974, or MDL for short) (1 ␮M) and semicarbazide (SCZ) (100 ␮M) for 4 h. Trypanocidal activity of the SSAO products H2 O2 and/or formaldehyde were also assayed at intervals during the 4 h incubation period. The survival of trypanosomes was determined using Alamar Blue (Raz et al., 1997) and untreated cells were used as a control. All experiments were carried out three times. 2.3. Hydrogen peroxides measurement H2 O2 generated during the oxidation of methylamine was determined using a continuous spectrophotometric method coupled to peroxidases (Holt et al., 1997). 4-Aminoantipyrine (400 nM) was added, which is oxidized by the hydrogen peroxide in samples, and then condenses with added vanillic acid (750 nM) to form a red dye with a maximum absorbance at 498 nm. A series of hydrogen peroxide concentrations were used as a standard. 3. Results and discussion SSAO mediated oxidation of methylamine produces one molecule each of formaldehyde, hydrogen peroxide and ammonia. T. brucei brucei (STIB 920) bloodstream forms were incubated

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at 37 ◦ C in HMI-11 medium with rVAP-1 protein or methylamine or both of them. As assessed by the Alamar Blue method, parasite viability was not affected by either rVAP-1 (1.5 ng ␮l−1 ) or methylamine (1 mM) alone, but parasites lost motility rapidly in the present of both (70% mortality after 1 h and no motile individual was found after 2 h) (Fig. 1). Further analysis demonstrated that the trypanocidal effect was methylamine-and concentrationdependent. Parasite death occurred when the concentrations of methylamine were higher than 1 mM in the presence of SSAO crude extract (Fig. 2A) or rVAP-1 (Fig. 2B). Therefore we proposed that cell death were triggered by SSAO/rVAP-1-mediated oxidation of methylamine. To test this hypothesis, we added SSAO inhibitors to prevent the oxidation. As we expected, parasites were protected from being killed by SCZ and MDL (Fig. 2A and B). H2 O2 was shown previously enough to kill trypanosomes in vitro (Ferrante et al., 1982, 1984; Rzepczyk et al., 1984) and formaldehyde is highly toxic to mammal cells (Gubisne-Haberle et al., 2004). Since these two highly toxic products formaldehyde and H2 O2 were both generated in the SSAO-/rVAP-1-mediated oxidation, we next determined which is primary for the antiparasitic effect. Parasites were treated with series concentration of H2 O2 and/or formaldehyde, or ammonia in vitro, 50% mortality was achieved by 100 ␮M of H2 O2 or 1 ␮M of formaldehyde or 500 ␮M of ammonia, respectively (Fig. 3A and B). This suggested that formaldehyde was more effective as much as 100 times than H2 O2 or 500 times than ammonia in trypanocidal capacity. To further prove that H2 O2 only has a limited effect during the process, catalase was added to remove H2 O2 from the reaction of rVAP-1 and methylamine. Catalase could easily rescue trypanosomes from H2 O2 toxicity (Fig. 3A), but was ineffective against the rVAP-1-mediated oxidation of methylamine (Fig. 4A), which produced a maximum of 0.5 ␮M H2 O2 and equivalent amount of formaldehyde in 2 h (Fig. 4B). All these results indicated formaldehyde was solely responsible for trypanocidal effects mediated by SSAO/rVAP-1. Previous studies demonstrated that polyamine oxidase (PAO) and xanthine oxidase (XO) killed trypanosomes in the presence of their substrates in vitro. H2 O2 -derived from PAO-spermine or spermidine and XO-xanthine system is the main factor acting against trypanosomes in Cape buffalo (Muranjan et al., 1997; Wang et al., 1999). In this study, we showed that SSAO/rVAP-1 displayed the same function as XO or PAO. However, formaldehyde from SSAO/rVAP-1’s oxidation of methylamine is a much more effective trypanocidal factor than H2 O2 . SSAOs play important physiological functions in mammals including humans (O’Sullivan et al., 2004). SSAO/rVAP-1 and their physiological substrates are enriched in the circulatory system of mammal hosts, where T. brucei mainly resides. The serum methy-

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Fig. 2. SSAO inhibitors block the trypanocidal activity of SSAO or rVAP-1. Parasites were cultured in medium with methylamine or methylamine and SSAO (A) or rVAP-1 (B) in presence or absence of SCZ (100 ␮M) and MDL (1 ␮M) for 4 h. () Methylamine + SSAO/rVAP-1 + MDL; () methylamine; () methylamine + SSAO/rVAP-1 + MDL; and (♦) methylamine + SSAO/rVAP-1.

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Fig. 3. Formaldehyde (HCHO) is much toxicity than hydrogen peroxide (H2 O2 ) and ammonia to T. brucei in vitro. Parasites were cultured in medium with hydrogen peroxide or formaldehyde or both of them, or ammonia for 4 h, respectively. (A) Catalase rescues trypanosomes from toxicity due to hydrogen peroxide but not from formaldehyde. () H2 O2 + catalase; () H2 O2 ; () HCHO + H2 O2 ; and (♦) HCHO. (B) Ammonia is less toxicity to parasites.

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Fig. 4. Formaldehyde is responsible for killing trypanosomes. (A) Catalase was added into medium with rVAP-1 and methylamine. () Methylamine; () methylamine + rVAP1 + catalase; and () methylamine + rVAP-1. (B) Hydrogen peroxide produced in the mixture of methylamine and SSAO/rVAP-1.

lamine concentration is estimated at ∼1 ␮M (Yu and Zuo, 1993). Theoretically, 1 ␮M methylamine produces an equivalent amount of formaldehyde, which could kill 50% of parasites in vitro (Fig. 3A). In this respect, it is theoretically possible that formaldehyde from methylamine could reach an effective concentration for partial killing T. brucei in vivo. However, it is unlikely that the steady serum concentration of methylamine is associated with an equivalent level of formaldehyde in vivo. Formaldehyde is difficult to detect since it immediately forms Schiff bases with amino groups in serum (e.g. epsilon amino group of lysine in proteins). Therefore the physiological significance of this finding remains an open question. As a matter of fact, it is interesting to know that low parasiteamia was found in the animals such as sheep, goat, buffalo and rabbit in which high serum SSAO activity was detected (Wang et al., 2000, Tyler et al., 2001; Li, 2006). In this study, we found T. brucei could be killed by SSAO mediated oxidation in vitro and we suggest that SSAO/rVAP-1 may play some unknown functions in non-specific defense of trypanosome infection in mammalian hosts. Therefore, it would be very interesting to note the effect of SSAO/rVAP-1 on trypanosome infection in animals and results may provide valuable data for better understanding the exact function of SSAO as innate trypanocide against trypanosomes. Acknowledgements We thank the Biotie Therapies, Turku, Finland kindly provided the recombinant humanVAP-1 (rVAP-1) and Dr. Peter Yu of the University of Saskatchewan provided the (E)-2-(4-fluorophenethyl)3-fluoroallylamine hydrochloride (MDL-72974A). We would also like to thank the anonymous reviewers who provided critical

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