Standard strains of S. aureus ATCC 29213 (Methicillin-sensible Staphylococcus aureus) and ATCC 33591 (Methicillin-resistant Staphylococcus aureus), and the methicillin-resistant clinical isolates Staphylococcus epidermidis 228 (MRSE) and Staphylococcus haemolyticus (225) presenting hetero-resistance to vancomycin and identified in previous study 15 were used. The clinical strains were isolated from bloodstream of patients from a tertiary hospital in Rio de Janeiro city, Brazil. The isolates of S. epidermidis were characterized as hetero-resistant to vancomycin through vancomycin agar screening test according to the National Comittee for Clinical Laboratory Standard (NCCLS) 16 and by evaluation of the population analysis profile (data not shown). All organisms were plated on 5% sheep blood agar base (Oxoid) at 35°C for 24 h.

BSC-40 cells from African green monkey kidney were propagated in Dulbeccos's modified Eagles's medium (DMEM; Invitrogen) supplemented with 8% calf serum, 2% heat-inactivated bovine serum (BRL/Gibco Laboratories), 50 μg mL^-1 gentamicin sulfate, 500 U mL^-1 penicillin, 100 μg mL^-1 streptomycin, 225 μg/mL sodium bicarbonate and 2,5 μg mL^-1 fungizon. Cells were grown as adherent cultures at 37°C in a 5% CO₂ incubator 17.

The naphthoquinones derivatives evaluated in this study (Figure 1) were obtained from the lapachol, which was isolated by extraction from T. avellanedae sawdust 18. The reactions were carried out in the laboratory of organic synthesis of the Natural Products Research Nucleus at UFRJ (NPPN-UFRJ). The α-lapachone, that we named as compound I was synthesized according to Hooker 19 and the other quinones (compounds II, II and IV) were synthesized according to Pinto and coworkers 20.

The MIC was evaluated by the dilution method in Mueller-Hinton broth medium (Oxoid), according to NCCLS 16, for each one of the naphthoquinones, with concentrations ranging from 2 to 512 μg/mL. Bacteria (10⁴ CFU/mL) were inoculated in the broth with the drug, and incubated at 35°C for 24 h.

MBC is the smaller concentration of the drug necessary for elimination of 99.9% of the microorganisms tested. The MBC was determined after the MIC assays. Tubes where the MIC results showed no bacterial growth, an aliquot of 0.1 mL was seeded in Mueller-Hinton agar without addition of drugs and the bacterial growth was evaluated for the MBC determination. After 24 h, at 35°C, if MIC = MBC or if MBC is one, two or three dilutions above of MIC, the drug is considered bactericide 21.

An overnight culture of S. aureus ATCC 33591 was diluted in BHI to 10⁷ CFU/mL and incubated for 30 min at 37°C. For labeling, cells were concentrated to 10⁸ CFU/mL in a methionine-free medium (MEM, Gibco) containing 200 μCi/mL of [³⁵S] methionine (Amershan) and subject to drug addition, compound IV or menadione (vitamin K3) 22 at final concentrations of 8, 16 and 32 μg/mL at 37°C, or heat treatment at 45°C 23. In all the tests, the cells were pulse-labeled for 30 minutes and collected by centrifugation at 12,000 g for 5 min. The cells were lysated with the addition of 40 ng/mL of lysostaphin (S. aureus) during 2 hours at 37°C. After incubation period, equal volumes of 0.5 M Tris-HCl (pH 7.2) buffer containing 4% SDS, 10% β-mercaptoethanol, 20% glycerol and 0.1 % bromophenol blue were added, and the samples were boiled for 5 min. Cellular extracts were subjected to Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. The gel was stained with Comassie-blue, destained, dried and exposed to X-ray films.

The neutral red assay is based on the incorporation of the supravital dye neutral red into living cells. Confluent monolayers of BSC-40 cells (96-well plate) were incubated with a specified concentration of the naphthoquinones for 24 h at 37°C. The control was performed in absence of drugs. Neutral red stock solution (0.1%) was prepared in deionized water and stored at room temperature. Before staining, a fresh 1:100 dilution of the dye was prepared. In accordance to Thompson (1998) 17, 100 μl/well of medium containing neutral red were added to living cells (50 μg/ml final concentration), and the microplates were incubated at 37°C in moist atmosphere with 5% CO₂ for 3 h. The cells were then washed with 4% formaldehyde and incubated at room temperature for 1 min. After formaldehyde discarding, methanol solution (50%) was added and incubated at room temperature for 20 min. The optical density at 490 nm was measured using a microtiter plate spectrophotometer. The uptake of neutral red is proportional to the number of viable (live) cells 21.

This test was performed according to Draize (1944) 24. Different concentrations of the naphthoquinones were prepared, according to Table 1. Ten healthy rabbits were selected for each drug solution and the animals separated for chamber adaptation 48 hours before the assay. The animals had not alimentary restrictions and periods of dark and light were intercalated in each 12 hours. The ambient temperature was maintained at 25 ± 2°C. The animals were depilated on the dorsal region 24 hours before the assay. The dorsal region was divided in two parts: the right side, with two limited areas with no blooding chases, and the left one, with two limited areas with intact skin. The concentrations of the alcoholic solutions of the naphthoquinones related to the MIC obtained (Table I) were applied on the pre-established limited areas of the animals. The compound IV was also tested in the concentration of 0.8 mg/mL (MIC 100×). The rabbits were in contact with the solution of the naphthoquinones during four hours, and observations were done during 24, 48, 72 and 96 hours. During this period, the appearance of inflammatory reactions (edema and/or erythematic areas) or any other toxic reactions due to the substances was evaluated. The signals and symptoms observed were classified in agreement with the Federal Hazardous Substances Act of the United States.

The MIC and MBC determination was performed to compare the antimicrobial effect of the naphthoquinones in MSSA and MRSA strains (S. aureus) and evaluate this effect in resistant coagulase-negative staphylococci (S. epidermidis and S. haemolyticus). The antimicrobial activity of the compounds against S. aureus (ATCC 29213 and ATCC 33591), S. epidermidis MRSE 228 and S. haemolyticus MRSH 225 isolates are shown in the Table 1. Compounds III and IV showed the best results, inhibiting the growth of all bacteria at concentration of 8 μg/mL. For the isolate S. epidermidis, the compound IV showed a MIC of 4 μg/mL. A MBC above 512 μg/mL was seen for all the compounds tested, indicating an antibacteriostatic activity.

To investigate a possible mechanism of action of naphthoquinones on the bacteria protein synthesis, the compound IV were selected to perform this analyze since it presented the lowest MIC. So, (±) 3-hydroxy-β-N-lapachone (compound IV) were added to the staphylococcal cells at different concentrations (8, 16 and 32 μg/mL) in the presence of ³⁵S-Met as described in Materials and Methods. After SDS-PAGE, the corresponding autoradiogram showed that this compound did not inhibit the bacterial protein synthesis at the tested concentrations but induced the expression of some proteins of 100, 70, 60 and 10 KDa (Figure 2). The same pattern of induction was observed when cells were submitted to 45°C. We have previously identified these proteins as heat-shock proteins (HSPs) also known as stress proteins 23. In order to determine if the cellular stress caused by the compound IV could be related to oxidative stress, the menadione (vitamin K3) 22, a well known oxidative stress agent, was tested at 8, 16 e 32 μg/mL, showing the same pattern of induction (data not shown). These results showed that the naphthoquinones analyzed cause a stress reaction in bacterial cell, suggesting that it could be related to oxidative stress.

The cytotoxicity was evaluated to determine the toxic concentration of these compounds and compare with the antibacterial concentration observed (MIC), for further analysis of their application in antimicrobial therapy. The compounds presented a considerable cytotoxicity. A concentration of 2 μg/mL of the compound IV was sufficient to kill 80% of the cell culture, while its minimal concentration to inhibit the bacteria was 8 μg/mL. It was observed that the other compounds are less toxic than compound IV. The precursor (compound I) did not present a severe toxicity to BSC-40 cells when compared to the other compounds used at the same concentration (Figure 3). The minimal inhibitory concentrations of the other substances tested with eukaryotic cells are listed in the Table 2. This effect on BSC-40 cells suggests that these compounds could be used in antineoplasic therapy or antibacterial therapy as topic preparations.

This assay was performed to determine the toxicity of these compounds (previously considerable a toxic substance after the cytotoxicity determination) in topic preparations. No damage was observed on the limited dermal areas of the animals evaluated at the periods of 24, 48, 72 and 96 hours after the application of naphthoquinones solutions in all concentrations used, including a concentration 100× higher than the MIC found for the compound IV (Table 3). Then, these compounds did not show dermal irritability when used in topic preparations.