Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98(6): 843-848, September 2003 843 Cytotoxicity and Potential Antiviral Evaluation of Violacein Produced by Chromobacterium violaceum CR Andrighetti-Fröhner, RV Antonio**, TB Creczynski-Pasa/***, CRM Barardi*, CMO Simões/+ Laboratório de Virologia Aplicada, Departamento de Ciências Farmacêuticas *Departamento de Microbiologia e Parasitologia **Laboratório de Bioquímica, Departamento de Bioquímica ***Laboratório de Bioenergética e Comunicação Celular, Universidade Federal de Santa Catarina, Campus Universitário Trindade, Florianópolis, SC, Brasil Natural products are an inexhaustible source of compounds with promising pharmacological activities including antiviral action. Violacein, the major pigment produced by Chromobacterium violaceum, has been shown to have antibiotic, antitumoral and anti-Trypanosoma cruzi activities. The goal of the present work was to evaluate the cytotoxicity of violacein and also its potential antiviral properties.The cytotoxicity of violacein was investigated by three methods: cell morphology evaluation by inverted light microscopy and cell viability tests using the Trypan blue dye exclusion method and the MTT assay. The cytotoxic concentration values which cause destruction in 50% of the monolayer cells (CC50) were different depending on the sensitivity of the method. CC50 values were ≥ 2.07 ± 0.08 µM for FRhK-4 cells: ≥ 2.23 ± 0.11 µM for Vero cells; ≥ 2.54 ± 0.18 µM for MA104 cells; and ≥ 2.70 ± 0.20 µM for HEp-2 cells. Violacein showed no cytopathic inhibition of the following viruses: herpes simplex virus type 1 (HSV-1) strain 29-R/acyclovir resistant, hepatitis A virus (strains HM175 and HAF-203) and adenovirus type 5 nor did it show any antiviral activity in the MTT assay. However violacein did show a weak inhibition of viral replication: 1.42 ± 0.68%, 14.48 ± 5.06% and 21.47 ± 3.74% for HSV-1 (strain KOS); 5.96 ± 2.51%, 8.75 ± 3.08% and 17.75 ± 5.19% for HSV-1 (strain ATCC/VR-733); 5.13 ± 2.38 %, 8.18 ± 1.11% and 8.51 ± 1.94% for poliovirus type 2; 8.30 ± 4.24%; 13.33 ± 4.66% and 24.27 ± 2.18% for simian rotavirus SA11, at 0.312, 0.625 and 1.250 mM, respectively, when measured by the MTT assay. Key words: violacein - cytotoxicity - antiviral - MTT assay - herpes simplex virus type 1- poliovirus - rotavirus hepatitis A virus - adenovirus During the last few years efforts have been made to increase the number of substances with antiviral activity. Few substances are known which provide an effective treatment of viral infections in vivo (Balfour 1999). Also, the therapeutic potency of most of the antiviral agents encountered so far is counterbalanced by their severe side effects in humans (Glatthaar-Saalmüller et al. 2001) and the efficacy of these drugs is limited by increases in viral resistance (Pillay & Zambon 1998, De Logu et al. 2000). The search for antiviral substances with high efficacy, low toxicity, and minor side effects therefore must continue. Natural products have been an abundant source of compounds which have proved useful in antiviral chemotherapy of infectious human diseases (Pujol et al. 1996, Bedoya et al. 2001) especially those originating from plant extracts and fermentation broths from soil bacteria, which provide compounds directly useful as drugs or as leads for the synthesis of new medicines (Nielsen 2002). This work received financial support from CNPq/MCT/Brazil. + Corresponding author. Fax: +55-48-331.9258. E-mail: Received 17 February 2003 Accepted 3 June 2003 Chromobacterium violaceum is a Gram(-) bacteria found in water samples and soils from tropical and subtropical regions of the world. Due to its biotechnological potential, C. violaceum had its genome sequenced by the Brazilian National Genome Project. The most notable characteristic of C. violaceum is the production of the chemically well characterized pigment named violacein (Bromberg & Duran 2001). Previous studies indicated antibiotic and antichagasic (Duran & Menck 2001), antitumoral (Melo et al. 2000), and antileishmanial (Leon et al. 2001) activities of violacein. The aim of this study was to assess the cytototoxicity and the potential antiviral activity of violacein against the viruses: Herpes Simplex Virus type 1 (HSV-1) strains KOS, 29-R/acyclovir resistant and VR733/ATCC; Poliovirus type 2 (PV-2); Simian rotavirus SA11 strain, Hepatite A virus (HAV) strains HAF203 and HM175 and Adenovirus type 5 (AdV-5), a respiratory strain. MATERIALS AND METHODS Compound - Violacein was isolated and purified from C. violaceum (CCT3496/JMC3496) as described by Duran et al. (1994). Violacein was dissolved in absolute ethanol (Merck) and 0.003% of dimethyl sulphoxide (DMSO, Merck) and stored at 4ºC protected from light until tested. The suitable dilutions for testing were made in cell culture medium as stated below and the stock solution was quantified by using a spctrophotometer (Pharmacia, Ultrospec 3000) at 577 nm. Cell culture and viruses - The cell lines used were 844 Antiviral Evaluation of Violacein • CR Andrighetti-Fröhner et al. Vero cells (Adolpho Lutz Institute, Brazil), HEp-2 cells (Biological Science Institute, University of São Paulo, Brazil), MA104 cells (Biological Science Institute, University of São Paulo, Brazil) and FRhK-4 cells (Macquarie University, NSW, Australia). All the cell lines were grown in 199 Medium (Sigma) supplemented with 10% fetal bovine serum (FBS - Gibco BRL), penicillin G (100 U/ml), streptomycin (100 µg/ml) and amphotericin B (0.025 µg/ml) (Gibco BRL). The cell cultures were maintained at 37ºC in a humidified 5% CO2 atmosphere. The following viruses were used: Herpes Simplex Virus type 1 (HSV-1) strains KOS and 29-R/acyclovir resistant (Laboratory of Pharmacognosy, Faculty of Pharmacy, University of Rennes, France); HSV-1 strain VR733 (American Type Culture Collection, Rockville, MA, US); Poliovirus type 2 (PV-2) - a vaccinal strain Sabin II (Adolpho Lutz Institute, Brazil); Simian rotavirus SA11 strain (RVSA11); Hepatitis A virus (HAV) strains HAF203 and HM175 (Federal University of Rio de Janeiro); and adenovirus type 5 (AdV-5) (Biological Science Institute, University of São Paulo, Brazil). HSV-1 strains and PV-2 were propagated in Vero cells; RV-SA11 was propagated in MA104 cells in the presence of trypsin (Sigma, 5 µg/ ml); AdV-5 was propagated in HEp-2 cells and HAV strains were propagated in FRhK-4 cells. Stock viruses were prepared as described previously (Barardi et al. 1998, Simões et al. 1999) and the supernatant fluids were harvested, titrated and stored at -80ºC until used. HSV-1 and AdV-5 titers were obtained by the limit-dilution method and expressed as 50% tissue culture infections dose per ml (TCID50/ml) (Reed & Müench 1938); PV-2 titer was performed by the plaque method (Burlenson et al. 1992) and expressed as plaque forming units (pfu/ml); HAV and RV-SA11 titers were performed by immunofluorescence assay and expressed as focus forming units per m (...truncated)