Toxicology Reports 3 (2016) 756762

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Toxicology Reports
journal homepage: www.elsevier.com/locate/toxrep

Cytotoxicity, hemolysis and in vivo acute toxicity of

2-hydroxy-3-anilino-1,4-naphthoquinone derivatives
Valeska Santana de Sena Pereira a , Cludio Bruno Silva de Oliveira a , Fernando Fumagalli b ,
Flvio da Silva Emery b , Naisandra Bezerra da Silva c , Valter F. de Andrade-Neto a,
a
Laboratory of Malaria and Toxoplasmosis Biology, Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, RN,
Brazil
b
Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeiro Preto, University of So Paulo, Ribeiro Preto, SP, Brazil
c
Laboratory of Histotecnology, Department of Morfology, Federal University of Rio Grande do Norte, Natal, RN, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The 1,4-naphthoquinones, important members of the family of quinones are used as both crude extracts
Received 4 July 2016 and as compound manipulated by the pharmaceutical industry. They have gained great emphasis by pre-
Received in revised form 29 August 2016 senting different pharmacological properties as antibacterial, antiviral, antiprotozoal and anthelmintic,
Accepted 15 September 2016
and has antitumor activity. Our aim was to evaluate the cytotoxicity, hemolytic activity and in vivo acute
Available online 16 September 2016
toxicity of three derivatives of 2-hydroxy-1,4-naphthoquinones. The cell viability in vitro against RAW
Cell Line displayed IC50 ranging of 483.52044.8 M, whereas in primary culture tests using murine
Keywords:
macrophages, IC50 were 315.81408.0 M for naphthoquinones derivatives 4a and 4c respectively,
Naphthoquinones
Cytotoxicity
besides no hemolysis was observed at the dose tested. The in vivo acute toxicity assays exhibited a
Hemolytic activity signicant safety margin indicated by a lack of systemic and behavioral toxicity up to 300 mg/kg, and
Acute toxicity at a dose of 1000 mg/kg the derivatives not triggering signs of toxicity although the compound 4a have
promoted hepatic steatosis and hyperemia in kidney tissue. Thereby, these modications decrease the
toxicity of the tested derivatives naphthoquinones, providing a high potential for the development of
news drugs.
2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction rash, fever, vomiting, diarrhea, abdominal pain, headache and,

occasionally, transaminase and amylase levels are abnormal [2].
The 1,4-naphthoquinones are widely distributed in nature and Despite several synthetic naphthoquinone derivatives, such as
may be used as crude extracts or as compounds manipulated by the the 2-acetoxy and 2-ethoxy-1,4-variants widely used in industry,
pharmaceutical industry [18,17]. These compounds have diverse the mechanisms involved in cytotoxicity of the quinone derivatives
pharmacological properties such as antimicrobial [46,49], antiviral are still largely unknown [47]. The cytotoxic effects of naphtho-
[8], antiprotozoals [32,33,35,45,10] and anthelmintic [19], besides quinoidal compounds such as menadione might be due to oxidative
of antitumoral activity [37]. stress and arylation of cellular thiols [39]. Hepatic biotransforma-
There are also already naphthoquinones reports against P. tion of napththoquinone derivates can lead to cell injury through
falciparum in vitro [24,15,19,12,44] and in vivo [35]; and the several mechanisms unknown yet [14]. Studies shown that the 2-
antiplasmodial activity of benzo [a] phenazine synthesized from hydroxy-1,4-naphthoquinone is a substance highly toxic, causing
1,2-naphthoquinone and beta-lapachone lapachol [3]. both hemolytic anaemia and renal tubular necrosis in rodent [26]
The atovaquone, a hydroxy-naphthoquinone which inhibits the and non-human primates models [22]
electron transport chain and pyrimidine biosynthesis in Plasmod- Previous studies by our group demonstrated antimalarial activ-
ium falciparum [48], it was used as a prophylactic agent for travelers ity in three novel naphthoquinoidal derivatives against rodent
[40]. It causes few side effects, the most common symptoms are malaria caused by P. berghei infected erythrocytes in mice [35].
According to Resolution CNS 251/97, pre-clinical research is the
rst step in the study on the development of new drugs and should
promote sufcient information as the possible therapeutic applica-
Corresponding author. tions and preview some risks with its use as toxicity to support the
E-mail address: [email protected] (V.F. de Andrade-Neto). conduct of research in humans [20]. Thus, in this study, we evalu-

http://dx.doi.org/10.1016/j.toxrep.2016.09.007
2214-7500/ 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
 V.S. de Sena Pereira et al. / Toxicology Reports 3 (2016) 756762 757

37 C, 20 l of MTT solution (5 mg/mL) were added to each well and,

after 3 h, the supernatant was removed and 200 l of DMSO were
added. The optical densities of the plates were read by a microplate
spectrophotometer (Thermo Plate ) at 570 nm. The IC50 for the pri-
mary cell culture and RAW cells was determined using GraphPad
PRISM software . The test was performed in three repeats.

2.3. Hemolysis assay

The test was performed as suggested by Rabelo et al. [34].

Compounds were tested at two concentrations each: 4a (100 and
500 mM), 4c (50 and 250 mM), and 4d (200 and 1000 mM). Ref-
erences to 100% and 0% hemolysis were made by incubating a
suspension of red cells with Triton X-100 1% (v/v) and 0.9% saline,
Fig. 1. Chemical structure of the derivatives of 2-hydroxy-3- respectively. The values tested were experimentally determined
anilino-1,4-naphthoquinone. 2-hydroxy-3-anilino-1,4-naphthoquinone from IC50 obtained in a test antiplasmodial, corresponding to 10
(4a), 2- hydroxy-3-(2-chloro-aniline)-1,4-naphthoquinone (4b),
and 50 times more than the respective IC50 . The hemolysis per-
2-hydroxy-3-(4-methoxy-aniline)-1,4-naphthoquinone (4c) and 2-hydroxy-
3-(2,6-dimethyl-aniline)-1,4-naphthoquinone (4d).
centage was calculated on the positive control group. Assays were
performed in triplicates and repeated twice.

ated the toxic potential of three derivatives naphthoquinones using

2.4. Acute toxicity
models in vitro and in vivo.
The acute toxicity was performed according protocol OECD
2. Materials and methods [30]. Groups of three female Swiss mice of 812 weeks weigh-
ing around 29.0 2 g were randomly separated into the following
2.1. Compounds groups: group I, 4a compound; group II, 4c compound; group III 4d
compound and group IV, untreated control. The compounds were
The tested 2-hydroxy-1,4-naphthoquinones derivatives were diluted in DMSO (maximum nal concentration of 4%) and admin-
synthesized according previously report [35], which are: 2- istered orally (per gavage), 200 l single dose. The negative control
hydroxy-3-anilino-1,4-naphthoquinone, 2-hydroxy-3-(2-chloro- group receiving DMSO 4%.
aniline)-1,4-naphthoquinone, 2-hydroxy-3-(4-methoxy-aniline)- As recommended, the initial dose tested was 300 mg/kg body
1,4-naphthoquinone and 2-hydroxy-3-(2,6-dimethyl-aniline)-1,4- weight, dose for starting the test if there is no information on the
naphthoquinone, designated 4a4d, respectively. (Fig. 1). The toxicity of the compound to be tested. As was observed no mor-
compound 4b showed problems with solubility thereby prevent- tality or toxicity signs, the test was repeated with a higher dose
ing the continuity of its use in this study. Compounds 4a, 4c and (1000 mg/kg body weight). Body weight of the animals was checked
4d were selected for the tests to be soluble in the solution with a on the day of administration of the compounds and on the 15th day
maximum of 1% DMSO and have satisfactory results in the screen- after administration, and the weight variation was calculated.
ing tests showing antimalarial activity when tested in a murine After treatment, the animals were observed dialy for 14 days.
model-Plasmodium berguei (Table 1). The signs observed were piloerection, changes in the eyes and
mucous membranes; physical changes associated with central ner-
2.2. Cytotoxicity assay vous, autonomic, cardiovascular and respiratory systems; pattern
of behavior and somatomotor activity, direct attention to tremors,
Cell viability was assessed by colorimetric MTT [3-(4,5- convulsions, salivation, diarrhea, lethargy, sleep and coma. After 14
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay, days, the animals were autopsied for some hispathological analy-
as described by Mosmann [23], with modications. The cells used sis. For dose 1000 mg/kg, the absence or presence of inammation
were murine macrophages obtained by washing the peritoneal cav- and its activity were determined in livers, spleen and kidneys of
ity from Swiss mice and Cell Line RAW 264.7. The compounds to each study group. The specimens were xed in 10% formaldehyde,
be tested were diluted with 0.1% DMSO in culture medium in serial dehydrated and embedded in parafn. Sections of 5 m thickness
dilution to obtain seven concentrations ranging 100015.6 g/mL were obtained for haematoxylin-eosin staining (H&E Easypath) and
(1:2; 7 dilution), corresponding to 377358.9 M for the 4a examined by light microscopy (40, Olympus BX50); additionaly,
derivative; 339053.0 M for derivative 4c and 341353.3 M for the tissues were stained with periodic acid Schiff (PAS) to indi-
4d. For tests, RAW 264.7 line cells (1 104 cells per well) and rectly to demonstrate area affected by steatosis due to dye has an
macrophages from primary culture cells (1 106 cells per well) afnity for glycogen in the liver sections and not by lipids. Fur-
were distributed into 96 well microplates. After 48 h incubation at thermore, the presence of degenerative lesions were examined:

Table 1
Antimalarial activity of three novel naphthoquinoidal derivatives measured by reduction of parasitaemia by Plasmodium berghei and survival of mice treated as compared to
untreated mice control.

Compound Parasitemia Parasitemia inhibition (%) Mean Survival (Days)

Day 5 Day 5 Day 5 Day 7

4a 0.7 2.2 53.3 18.5 32.5 6.6

4c 0.5 2.0 66.6 25.9 26.0 4.0
4d 1.5 2,7 0 0 26.7 2.5
Vehicle (control) 1.5 2.7 0 0 20 2.7

Compounds previously tested; adapted from [35].

758 V.S. de Sena Pereira et al. / Toxicology Reports 3 (2016) 756762

Table 2
IC50 (M) of RAW 264.7 cells and murine resident peritoneal macrophages (RPM) exposured to concentration serial dilution of naphthoquinones derivatives.

Compounds RAW 264.7Cells RPMa

2-hydroxy-3-anilino-1,4-naphthoquinone (4a) 483,5 195.40 315,8 31.30

2-hydroxy-3-(4-methoxy-aniline)-1,4-naphthoquinone (4c) 714,9 182.95 532,6 103.24
2-hydroxy-3-(2,6-dimethyl-aniline)-1,4-naphthoquinone (4d) 2044,8 93.99 1408,0 52.49

Results show the mean of IC50 values SD (concentration required to inhibit cell growth by 50%) in micromolar. Data represent the means of three independent experiments,
with each concentration tested in triplicate.
a
Resident peritoneal macrophages.

Fig. 3. Percent hemolysis in vitro naphthoquinone of the 4a, 4c e 4d derivatives. The

values tested there were 10 and 50 times higher than the IC50 obtained in antiplas-
modial testing. Data are expressed as mean standard deviation. Was used ANOVA
followed by Tukeys test. The average followed by different letters are statistically
different (p < 0.01).

Table 3
Signs of acute toxicity and mortality derivatives naphthoquinones 4a, 4c and 4d.

Parameters observed Observations

300 mg/kg 1000 mg/kg

Fig. 2. Doseresponse curves of RAW 264.7 cells (A) and Resident peritoneal
macrophages (B), after treatment for 24 h with 2-hydroxy-1,4-naphthoquinones Skin/Fur W.C. W.C.
derivatives. The concentrations ranging between 0.016 to 1,0 mg/mL. Results are Eyes and Mucous Membranes W.C. W.C.
expressed in percentage of control. Three independent experiments are performed Cardiac/Respiratory Signs W.C. W.C.
in triplicate. Behaviour Pattern W.C. W.C.
Somatomotor Activity W.C. W.C.
Salivation N.O. N.O.
Tremors N.O. N.O.
vacuolar, micro and macro- vesicular steatosis and the presence Convulsions N.O. N.O.
or absence of brosis and necrosis [43,13]. Lethargy N.O. N.O.
The animal tests were approved by the Animal Ethics Committee Sleep N.O. N.O.
(CEUA/UFRN) under the protocol number 046/2013. Coma N.O. N.O.
Mortality N.O. N.O.
Necropsy W.C. macroscopic W.C. macroscopic
2.5. Statistical analyses W.C.: Without Change; N. O.: Not observed.

The IC50 was estimated by linear interpolation, in compar-

At the doses tested, none of the compounds caused alteration
ison with untreated controls, using software HN-NonLin V1.1
of behavior or have led to symptoms related to the central ner-
[29]. For the acute toxicity test, the results were expressed in
vous system, autonomic, circulatory and/or respiratory during the
mean standard deviation. The change of data between the treated
period tested as summarized in Table 3.
groups and controls was carried out by applying the Simple Anal-
During animal necropsy observed no macroscopic change of
ysis of Variance (ANOVA) followed by Tukey test (95% condence
organs for both compounds in the two tested doses as well as for
interval), using the statistical program Assistat 7.7 beta [42]. The
the untreated control.
results were considered statistically signicant for p-value 0.05.
The weight of the animals decreased in a dose-dependent man-
ner in animals treated with the naphthoquinones derivatives,
3. Results but at a dose of 1000 mg/kg there was no signicant difference
between the treated groups and the untreated control and to dose
The IC50 values for 2-hydroxy-1,4-naphthoquinones deriva- of 300 mg/kg only derivative 4c gained less weight than the other
tives ranging from 483.5 to 2044.8 M for RAW 264.7 cells and groups (Fig. 4).
from 315.8 to 1408.0 M for macrophages from primary cul- The Fig. 5 displays the weight of the organs of the animals treated
ture cells. 4d compound presented lower toxicity (Detailed data at dose of 1000 mg/kg. Only the group average 4c liver weight was
on Table 2). The viabilities of cells exposed to 2-hydroxy-1,4- higher compared to untreated control. Nor spleen or kidneys of any
naphthoquinones derivatives were declined with the increase of groups were statistically different from the control group.
concentration (Fig. 2). None of the compounds caused hemolysis at None injury was observed in the liver from animals of the group
the doses tested (Fig. 3). control, hepatocytes with cytoplasm, nucleus, nucleolus presented
 V.S. de Sena Pereira et al. / Toxicology Reports 3 (2016) 756762 759

increased levels of hydrogen peroxide and oxidized glutathione,

and react with tissue nucleophiles to modify proteins covalently
[28,18,6,12].
Ours results in cytotoxicity assays show that the derivatives
have low toxicity to the two cell types tested when compared
to other studies. Salustiano et al. [38] tested pentacyclic 1,4-
naphthoquinones on peripheral blood mononuclear cells (PBMCs)
and found the IC50 ranging from 8.56 to 23.50 M; Kishore et al.
[17] tested seven compounds on PBMCs and the compounds 8-
Fluoro-5-hydroxy-7-methyl-1,4-naphthoquinone (188,7 M) and
2,5-Dihydroxy-7-methyl-1,4-naphthoquinone (54 M) were the
least toxic, whilst the other compounds including 5-Hydroxy-7-
Fig. 4. Weight gain of mice treated with naphthoquinone derivatives 4a, 4c and 4d methyl-1,4-naphthoquinone (7-MJ) (18.4 M) were more toxic. In
and the untreated control. The change in weight was dose-dependent and dose of a study of six naphthoquinones compounds to HL-7702, Guo et al.
1000 mg/kg there was no signicant difference between groups. Data are expressed [14] observed that 2-hydroxy-1,4-naphthoquinone was less toxic.
as mean standard deviation (3 animals). Was used ANOVA followed by Tukeys The low toxicity of the compounds can be explained by low liposol-
test. The average followed by different letters are statistically different (p < 0.05).
ubility of 2-hydroxy-1,4-naphthoquinone which ensures that its
cell penetration is relatively poor [14].
Another good method for cytotoxicity evaluation is hemoly-
sis, which is characterized by the erythrocyte rupture with release
of hemoglobin. This, as will be free in plasma causes damage to
various vital organs such as the liver, kidney and heart. Thus, it
is necessary to observe the hemolytic activity in the screening of
biological and toxicological activities of plant extracts and deriva-
tives [5,9]. Furthermore, the lysis of red blood cells prevents direct
intravenous administration of desired agents and often increases
the toxicity of these agents when administered by other routes
[7]. Many studies of naphthoquinones derivatives reported that
these cause hemolytic anaemia in vivo [21,4,25,26,28]. Hemolysis
induced naphthoquinones has been attributed to the formation of
Fig. 5. Weight of spleen, kidney and liver of mice treated with naphthoquinone reactive oxygen species from the redox-cycling process induced by
derivatives orally administered (dose 1.000 mg/kg) and untreated control. Data are naphthoquinones [28]. None of the derivatives naphthoquinones
expressed as mean standard deviation. Was used ANOVA followed by Tukeys test. tested showed hemolytic activity at concentrations tested, indicat-
The average followed by different letters are statistically different (p < 0.01).
ing that the scaffold of naphthoquinoidal compounds used in our
work is less toxic than those previously described in the literature.
normal aspects and intact central vein. However, in the treated The animal body weight is an important factor to evaluate
groups with the compounds 4a, 4c and 4d an inammatory inl- the toxicity of a substance [16]. In our study, for the dose to
trate and congested blood vessels were observed. In the group 1000 mg/kg there was no signicant difference in weight gain
treated with 4a compound was also observed hemorrhagic foci and between the treated and the untreated group. However, at the dose
steatosis (Fig. 6). Using PAS staining, can be observed the circum- of 300 mg/kg, the compound 4c whose radical contains a methoxy
scription of injury due to steatosis (Fig. 7). group, presented less weight than the untreated group, but not
Only the group treated with the compound 4a showed signs statistically different from the other treatment groups.
of nephrotoxicity, which was observed by the presence of tissue Changes in organ weight have been accepts as indicators of
hyperemia (Fig. 8). No microscopic changes were observed in the test-induced changes, which are often associated with treatment-
spleen of the animals treated and untreated animals (data not related effects [41]. Regarding the weight change of organs, again
shown). only 4c group presented changes, whose liver had greater weight
gain compared to the other groups, although not being the group
4. Discussion most hepatotoxicity by histological analysis of organs.
In acute toxicity tests all derivatives exhibited a signicant mar-
The naphthoquinone derivatives are toxic and several stud- gin of safety, proven by the absence of behavioral and systemic
ies have shown that the structure changes in the molecule toxicity in the tested dose of 300 mg/kg, ie, there were no adverse
have reduced its cytotoxic effect, and improve biological activ- effects related to animals. Unable to perform the assay at a con-
ity. Davanco et al. [9], evaluated a prodrug primaquine and when centration of 2000 mg/kg because the compounds are non-polar in
compared with the primaquine, it was less cytotoxic to BGM and nature, it is not possible dilution in water in high concentrations.
HepG2 cells, caused less hemolysis of G6PD decient red blood The test was then conducted with a dose of 1000 mg/kg, which
cells and caused less alteration in the biochemical parameters. In were not observed signs of toxicity. Thus, one may suggest the
their study Oliveira et al. [31] produced a derivative of vanillin classication of compounds in category 4 of GHS (Globally Har-
molecule from its condensation with resorcinol and absence of monized System of Classication and Labelling of Chemicals) [30].
cytotoxicity observed in murine macrophage cells derived com- A necropsy of the animals at the two doses tested showed macro-
pound, while the vanillin presented an IC50 of 645 g/mL. This scopic aspects of normal organs.
toxicity can be explained in two ways: by one-electron reduction, The liver, kidney, and spleen were removed for histopathologi-
forming semiquinones that self-oxidize quinone, with production cal analysis because they are among the organs primarily affected
of active oxygen species, or by reaction with cellular nucleophiles, by metabolic reaction caused by toxicant [11]. The liver is the
due to its characteristic electrophilic [26,18]. This is because the main target organ affected where exposed to toxin substances, after
1,4-naphthoquinones can catalyze redox cycling to produce reac- being absorbed in intestines and metabolized to other compounds
tive oxygen species, generate a highly oxidative environment with [36]. In our study, the ndings revealed less severe morphologi-
760 V.S. de Sena Pereira et al. / Toxicology Reports 3 (2016) 756762

Fig. 6. Analysis representative histological sections of the livers of mice treated with derivatives naphthoquinones orally administered (dose 1.000 mg/kg) and untreated
control. The arrowheads indicate inammatory inltrates. Asterisks indicate congested vessels. The square indicates steatosis. (Hematoxylin & Eosin, magnication 400).

Fig. 7. Histological sections stained, with periodic acidschiff (PAS) method, of the livers of mice treated with derivatives naphthoquinones orally administered (dose
1.000 mg/kg) and untreated control. In the image, the circles stand one hepatocyte. Black arrows point the nucleus of hepatocytes and large arrows show the glycogen. While
accumulation of lipids doesnt show a positive reaction the method; hashtags (4a) show the lipid droplets indicating steatosis. The asterisk shows a congested vessel and the
arrow head indicates inammatory inltrate.

cal changes in liver of mice treated, being observed only points of Second Munday et al. [28], beyond the hemolytic activity,
inammatory inltrate and congested vessels. Derivative 4a was many naphthoquinone derivatives can cause necrosis of tubular
the one who presents steatosis, proving to be more hepatotoxic epithelial cells, the substituents at C-3 of position 3 of the 2-
than the other compounds. hydroxy and 2-amino-1,4-naphthoquinone reduces or eliminates
 V.S. de Sena Pereira et al. / Toxicology Reports 3 (2016) 756762 761

Fig. 8. Histological analysis of representative sections of the kidney of mice treated with derivatives naphthoquinones orally administered (dose 1.000 mg/kg) and untreated
control. The arrowheads indicate bleeding tissue (Hematoxylin & Eosin, magnication 400).

the nephrotoxicity, though the mechanism of how this occurs Acknowledgements

remains unknown. Contrary to what was observed in our study, in
which only the 4a group exhibited signs of kidney damage, proven The authors would like to recognize nancial support received
by observation of tissue hyperemia, several studies have shown kid- from CNPq (476637/2012-0). VFAN (301837/2012-0) is CNPq/PQ-
ney damage, with tubular necrosis in rats treated with derivatives Research Productivity Fellowship recipients.
of hydroxy derivatives such as 2-hydroxy-1,4-naphthoquinone
[25], 2-hydroxy-3-alkyl-1,4-naphthoquinone [26] and 2-amino-
1,4-naphthoquinone [27]. Furthermore, no changes in the renal Appendix A. Supplementary data
tissue of the treated groups with a naphthoquinone derivative was
observed, the glomeruli and capsules appeared normal and the Supplementary data associated with this article can be found,
Bowmans space are also marked clearly. in the online version, at http://dx.doi.org/10.1016/j.toxrep.2016.09.
The hematopoietic system is very sensitive to toxic compounds 007.
and serves as an important index of the physiological and patholog-
ical status in both animals and humans [1]. Histological analysis of
the spleen showed an apparently normal tissue, with morphologi- References
cally distinct compartments, white and red pulp distinguishable.
[1] A.A. Adeneye, et al., Preliminary toxicity and phytochemical studies of the
Compared with the results found by Munday et al. [25] stem bark aqueous extract of Musanga cecropioides in rats, J. Ethnopharmacol.
with 2-hydroxy-1-4-naphthoquinone and by Munday et al. [28] 105 (2006) 374379.
with 2-hydroxynaphthoquinone derivatives, which showed severe [2] H.O. Alkadi, Antimalarial drug toxicity: a review, Chemotherapy 53 (2007)
385391.
hemolytic anaemia, reected by splenic enlargement, besides the
[3] V.F. Andrade-Neto, et al., Antimalarial activity of phenazines from lapachol,
renal tubular necrosis, our compounds have reduced toxicity, beta-lapachone and its derivatives against Plasmodium falciparum in vitro and
can be a result of the anilino group at C-3 of naftoquinoidal Plasmodium berghei in vivo, Bioorg. Med. Chem. Lett. 14 (5) (2004) 11451149.
[4] S. Ansbacher, et al., Toxicity of menadione, menadiol and esters, J. Pharmacol.
ring.
Exp. Ther. 75 (1942) 111124.
[5] V.O. Bednarczuk, et al., Testes in vitro e in vivo utilizados na triagem
toxicolgica de produtos naturais, Viso Acadmica, Curitiba, 2010, pp. 11.
5. Conclusion [6] Belorgey, et al., 1,4-Naphthoquinones and others NADPH-dependent
glutathione reductase-catalyzed redox cyclers as antimalarial agents, Curr.
Pharm. Des. 19 (14) (2013) 25122528.
In our work, the 2-hydroxy-3-anilino-1,4-naphthoquinone [7] H.T. Chen, et al., Cytotoxicity, hemolysis, and acute in vivo toxicity of
derivatives do not show severe toxicity in vitro and in vivo mod- dendrimers based on melamine, candidate vehicles for drug delivery, J. Am.
els, none clinical serious sign were observed in treated animals. Chem. Soc. 126 (2004) 1004410048.
[8] E.C. Da Costa, et al., Synthetic 1,4-pyran naphthoquinones are potent
The only sign of toxicity observed for all products was hepatotox- inhibitors of dengue virus replication, PLoS One 8 (12) (2013) e82504.
icity, which may be explained by the extremely high dose. Thus, [9] M.G. Davanco, et al., Evaluation of antimalarial activity and toxicity of a new
the chemical structure of these compounds is closed related to the primaquine prodrug, PLoS One 9 (2014).
[10] M.V. De Arajo, et al., Synthesis, leishmanicidal activity and theoretical
toxicity of naphthoquinones which are compounds having known evaluations of a series of substituted bis-2-hydroxy-1,4-naphthoquinones,
toxicity. Molecules 19 (9) (2014) 180195.
762 V.S. de Sena Pereira et al. / Toxicology Reports 3 (2016) 756762

[11] E. Dybing, et al., Hazard characterization of chemicals in food and diet: dose [30] OECD, Guidelines 423, Acute Oral Toxicity Acute Toxic Class Method, in
response, mechanism and extrapolation issues, Food Chem. Toxicol. 42 (2002) OECD Guidelines for Testing of Chemicals, OECD, Paris, France, 2001, pp. 114.
237282. [31] C.B.S. Oliveira, et al., Comparative study on the antioxidant and
[12] K. Ehrhardt, et al., The antimalarial activities of methylene blue and the anti-toxoplasma activities of vanillin and its resorcinarene derivative,
1,4-naphthoquinone 3-[4-(triuoromethyl)benzyl]-menadione are not due to Molecules 19 (2014) 58985912.
inhibition of the mitochondrial electron transport chain, Antimicrob. Agents [32] S. Pieretti, et al., Naphthoquinone derivatives exert their antitrypanosomal
Chemother. 57 (5) (2013) 21142120. activity via a multi-target mechanism, PLoS Negl. Trop. Dis. 7 (2013).
[13] D.C.P. Franco, et al., Early cytotoxic and genotoxic effects of atrazine on Wistar [33] E.G. Pinto, et al., Potential of
rat liver A morphological, immunohistochemical, biochemical, and molecular 2-hyroxy-3-phenylsulfanylmethyl-[1,4]-Naphthoquinones leishmania (L.)
study, Ecotoxicol. Environ. Saf. 78 (2012) 170177. infantum: biological activity and structure-activity relationships, PLoS One 9
[14] J. Guo, et al., Study on cytotoxicity and structureactivity relationship of (8) (2014).
HL-7702 cell exposed to naphthoquinones, Environ. Toxicol. Pharmacol. 33 [34] L. Rabelo, et al., A lactose-Binding lectin from the marine sponge Cinachyrella
(2012) 408413. apion (Cal) induces cell death in human cervical adenocarcinoma cells, Mar.
[15] H. Hussain, et al., New quinoline-5,8-dione and hydroxynaphthoquinone Drugs 10 (2012) 727743.
derivatives inhibit a chloroquine resistant Plasmodium falciparum strain, Eur. [35] L.C.D. Rezende, et al., In vivo antimalarial activity of novel
J. Med. Chem. 54 (2012) 936942. 2-hydroxy-3-anilino-1,4-naphthoquinones obtained by epoxide ring-opening
[16] A.I. Jahn, P.K.H. Gnzel, The value of spermatology in male reproductive reaction, Bioorg. Med. Chem Lett. 23 (6) (2013) 45834586.
toxicology: do spermatologic examinations in fertility studies provide new [36] H. Rhiouania, et al., Acute and subchronic toxicity of an aqueous extract of the
and additional information relevant for safety assessment? Reprod. Toxicol. leaves of Herniaria glabrain rodents, J. Ethnopharmacol. 118 (2008) 378386.
11 (1997) 171178. [37] K. Salomo, et al., Trypanosoma cruzi mitochondrial swelling and membrane
[17] N. Kishore, et al., Cytotoxicity of synthesized 1,4-naphthoquinone analogues potential collapse as primary evidence of the mode of action of
on selected human cancer cell lines, Bioorgan. Med. Chem. 22 (2014) naphthoquinone analogues, BMC Microbiol. 3 (2013) 13196.
50135019. [38] E.J.S. Salustiano, et al., Comparison of the cytotoxic effect of lapachol,
[18] Y. Kumagai, et al., The chemical biology of naphthoquinones and its -lapachone and pentacyclic 1,4-naphthoquinones on human leukemic cells,
environmental implications, Annu. Rev. Pharmacol. 52 (2012) 221247. Invest. New Drugs 28 (2010) 139144.
[19] D.A. Lanfranchi, et al., Synthesis and biological evaluation of [39] N. Sata, et al., Menadione induces both necrosis and apoptosis in rat
1,4-naphthoquinones and quinolone-5,8-diones as antimalarial and pancreatic acinar AR4-2J cells, Free Radic. Biol. Med. 23 (6) (1997) 844850.
schistosomicidal agents, Org. Biomol. Chem. 10 (2012) 63756387. [40] P. Schlagenhauf, E. Petersen, Malaria chemoprophylaxis: strategies for risk
[20] Ministrio da Sade, Resoluco CNS 251/97 Normas de Pesquisa com Novos groups, Clin. Microbiol. Rev. 21 (2008) 466472.
Frmacos, medicamentos, Vacinas e Testes Diagnsticos Envolvendo Seres [41] R.S. Sellers, et al., Society of toxicologic pathology position paper: organ
Humanos, Dirio Ocial da Unio 1997, Brasil, 1997, pp. 21117. weight recommendations for toxicology studies, Toxicol. Pathol. 35 (2007)
[21] H. Molitor, H.J. Robinson, Oral and parenteral toxicity of vitamin K1 phthiocol 751755.
and 2 methyl 1,4-naphthoquinone, Proc. Soc. Exp. Biol. Med. 43 (1940) [42] F.A.S. Silva, C.A.V. Azevedo, Principal components analysis in the software
125128. assistat-statistical attendance World Congress On Computers In Agriculture,
[22] R.K. Cooney Morrison, et al., Oral toxicology studies with lapachol, Toxicol. vol. 7, American Society of Agricultural and Biological Engineers, Reno, NV,
Appl. Pharmacol. 17 (1970) 1. USA, 2009.
[23] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: [43] P.J. Scheuer, Classication of chronic viral hepatitis: a need for reassessment,
application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 J. Hepatol. 13 (1991) 372374.
(1983) 5563. [44] N.B. Souza, et al., Blood shizonticidal activities of phenazines and
[24] T. Mller, et al., Glutathione reductase-catalyzed cascade of redox reactions to naphthoquinoidal compounds against Plasmodium falciparum in vitro and in
bioactivate potent antimalarial 1,4-naphthoquinonesa new strategy to mice malaria studies, Mem. Inst. Oswaldo Cruz 109 (5) (2014) 546552.
combat malarial parasites, J. Am. Chem. Soc. 133 (2011) 1155711571. [45] F. Souza-Silva, et al., Evidences for leishmanicidal activity of the
[25] R. Munday, et al., Haemolytic activity and nephrotoxicity of naphthoquinone derivative epoxy--lapachone, Exp. Parasitol. 147 (2014)
2-hydroxy-1,4-naphthoquinone in rats, J. Appl. Toxicol. 11 (1991) 8590. 8184.
[26] R. Munday, et al., Comparative toxicity of [46] T. Sreelatha, et al., Synthesis and SAR study of novel anticancer and
2-hydroxy-3-alkyl-1,4-naphthoquinones in rats, Chem. Biol. Interact. 98 antimicrobial naphthoquinone amide derivatives, Bioorg. Med. Chem Lett. 24
(1995) 185192. (15) (2014) 36473651.
[27] R. Munday, et al., Effect of inducers of DT-diaphorase on the haemolytic [47] J.S. Sun, et al., Menadione-induced cytotoxicity to rat osteoblasts, Cell. Mol.
activity and nephrotoxicity of 2-amino-1,4-naphthoquinone in rats, Chem. Life Sci. 53 (1997) 967976.
Biol. Interact. 155 (2005) 140147. [48] A.B. Vaidya, et al., Structural features of Plasmodium cytochrome b that may
[28] R. Munday, et al., Structure-activity relationships in the haemolytic activity underlie susceptibility to 8-aminoquinolines and hydroxynaphthoquinones,
and nephrotoxicity of derivatives of 1,2- and 1,4-naphthoquinone, J. Appl. Mol. Biochem. Parasitol. 58 (1993) 3342.
Toxicol. 27 (2007) 262269. [49] J.Y. Yang, H.S. Lee, Antimicrobial activities of active component isolated from
[29] H. Noedl, Non Linear Evaluation of Malaria Drug Sensitivity Data Lawsonia inermis leaves and structure-activity relationships of its analogues
(HN-NonLinV1.1), Armed Forces Research Institute for Medical Sciences, against food-borne bacteria, J. Food Sci. Technol. 52 (4) (2013) 24462451.
Bangkok, Thailand, 2002 http://www.meduniwien.ac.at/user/harald.noedl/
malaria/software.html.