Dietary Psidium guajava Supplementation Reducing Ferric Nitrilotriacetate Toxicity in Puntius altus
The efficiency of Psidium guajava was studied to illustrate the reduction of ferric nitrilotriacetate (Fe-NTA) toxicity in Puntius altus via the histopathology analysis. The fish (n = 40) were randomly divided into four groups. Each fish was transferred to each aquarium as follows: G1 and G2 were obtained normal fish food; G3 and G4 were obtained guava leaf extract 60 mg g-1 fish food. After 28 days dietary supplement, fish in G2 and G4 were injected intraperitoneal of 9 mg Fe kg-1 b.wt. Twenty-four hour after injection, lesions were especially most evident in the G2. The gills were observed epithelial lifting, lamellar cell hyperplasia. Blood congestion was seen in sinusoids and hepatocytes necroses were also observed. Renal tubular swelling and necrosis were seen. Some areas were found hemosiderin pigment accumulation. Fish with guava pre-treatment (G4) showed slightly alteration when compare those of G2 group. The results suggested that P. guajava leaf extract pre-obtained may play an important role in the reduction of Fe-NTA toxicity in fish.
February 23, 2011; Accepted: April 16, 2011;
Published: June 25, 2011
Thailand is considered one of the countries which bring medical plants into
use and sometimes they refer to it as Thai Traditional Medicine (TTM)
which can be used to treat humans and animal diseases. Ascorbic Acid (AA) is
an essential vitamin for normal growth and physiological functions in animals
including fishes. Most teleosts are unable to synthesize AA because of the lack
of L-gulonolactone oxidase (Fracalossi et al., 2001).
Therefore, an exogenous source of AA is required in fish diets. It functions
as a general water-soluble reagent, on collagen formation, iron metabolism and
the response to stress (Vijayavel et al., 2006).
Many authors have reported the efficacy of AA in reducing genotoxicity in Oreochromis
niloticus induced by lead (Jiraungkoorskul et al.,
2008); Poronotus triacanthus induced by copper (Jiraungkoorskul
and Sahaphong, 2007) and Puntius altus induced by cadmium (Jiraungkoorskul
et al., 2007), using the micronucleus and nuclear abnormality tests.
It has also been reported the efficacy of AA in reducing the histopathological
alterations in fish after cadmium exposure (Jiraungkoorskul
et al., 2006). Edema, lamellar cell hyperplasia, epithelial lifting
and aneurysm were observed in the gills. There were blood congestion in sinusoids,
vacuolation of hepatocytes, hemosiderin accumulation, apoptosis and nuclear
pyknosis. Glomerulus atrophy, hydropic swelling, hyaline casts and necrosis
were seen. Fortunately, in the combination of cadmium and AA treated group,
they showed similar alterations as those observed in the cadmium treated alone
group but they were less severe. The findings of this study can be used as guidelines
for developing programs to help the fish which are cultured near heavy metal
contaminated areas (Jiraungkoorskul et al., 2006).
However, the uses of natural origin are preferred over the others because they
are safe and non-toxic and have no resistance problems.
Psidium guajava Linn. (Farang in Thai, or Guava
in English) constituents include ascorbic acid, triterpenes (Begum
et al., 2004), carotenoids (Mercadante et al.,
1999) and flavonoids (Rattanachaikunsopon and Phumkhachorn,
2007). The leaves are elliptic to ovate about 5-15 cm in length. The flowers
are white, with five petals and numerous stamens. This plant produces edible
round or pear shaped fruit which contains high amounts of calcium. Guava leaves
are used for medicinal purposes, as a remedy to diarrhea. Recent studies prove
that guava has sugar lowering properties to help diabetics lower their sugar
count (Gutierrez et al., 2008). In addition, crushed
leaves from guava plants can be applied on wounds, ulcers and rheumatic places
and toothache can be relieved by chewing the leaves. Lastly, leaves extracts
can also have antimicrobial properties and can also be for coughs (Jaiarj
et al., 1999). The study survey revealed that there are no scientific
studies carried out regarding the efficiency of this plant in fish. Ferric nitrilotriacetate
(Fe-NTA) has been chosen as a model organic compound to study the protective
effect of this herb in its toxicity. Awai et al. (1979)
first reported glycosuria and hepatic parenchymal iron deposits in rats following
intraperitoneal injections with Fe-NTA. Its toxicity is assumed to be caused
by the elevation of free serum iron concentration, following its reduction at
the luminal side of the proximal tubule (Liu et al.,
1991), generating reactive oxygen species, leading to lipid peroxidation
and induce oxidative stress in liver (Iqbal et al.,
1995). This present study was designed to assess the ability of the extract
from P. guajava against ferric nitrilotriacetate induced toxicity by
using histopathological analysis.
MATERIALS AND METHODS
Animal model: This study was performed at the Department of Pathobiology,
Faculty of Sciences, Mahidol University, Bangkok, Thailand in 2010. Red tailed
tinfoil barb, P. altus, 16.76±1.72 g in b.wt. and 9.85±0.50
cm in total length, were purchased from a commercial hatchery in Bangkok, Thailand.
Tap water was filtered with activated charcoal to eliminate chemical contamination.
The physicochemical characteristics of water were measured daily, according
to the experimental procedures described in Standard Methods for the Examination
of Water and Wastewater (American Public Health Association,
2005). Under laboratory condition, fish were acclimated for 30 days at 28.5±1.0°C,
pH = 6.8-7.0, total hardness = 70-80 mg L-1 (as CaCO3),
alkalinity = 75-80 mg L-1 and conductivity = 190-210 μmhos cm-1.
A 16:8 h light-dark cycle was maintained throughout. Chlorine residual and ammonia
were below detection limits. Fish were fed twice a day with 37%-protein commercial
fish food (Charoen Pokphand Group, Bangkok, Thailand). The quantity of food
was 2% of the initial body weight per day. The animal care and handling in this
research was performed following the instruction of the Mahidol University-Institutional
Animal Care and Use Committee (MU-IACUC). Therefore, this research was followed
the mammal animal care and use i.e., (1) Use, care and transportation of fish
for toxicopathological testing was complied with all applicable animal welfare
laws. (2) Number of fish was kept to the minimum requirement for achieve scientifically
valid results. (3) All protocols were taken to avoid the discomfort, distress
or pain in the fish. (4) The appropriate dosage of the anesthesia was 200 mg
L-1 ethyl-3-aminobenzoate methanesulfonate salt (MS222, Sigma) and
the euthanasia was overdose of this chemical.
Preparation of dry leaf with fish food: Guava was collected from local
area in Nakorn Prakom Province, Thailand. Fresh leaves were washed several times
in water, dried at 45°C for 72 h and made semi powder by crushing using
a mortar and pestle. The extraction was done by following the method of Winkaler
et al. (2007) with slight modifications. Fish food was grounded in
a blender and hydrated with 0.7 mL g-1 distilled water, mixed with
the leaf semi powder extract and extruded through a minced-meat processing machine.
Later, the mixture was broken into small pellets by hands and dried at 60°C
for 48 h. The fishes were fed twice a day (2% b.wt.) with the prepared dry leaf
food supplementation within 28 days.
Preparation of Ferric Nitrilotriacetate (Fe-NTA) solution: A solution
of Fe-NTA was prepared by the method of Awai et al.
Experimental design: The fish (n = 40) were randomly divided into four groups. Each fish group was transferred to each aquarium as follows: G1 and G2 were obtained normal fish food; G3 and G4 were obtained guava leaf extract 60 mg g-1 fish food. After 28 days dietary supplement, fish in G2 and G4 were injected intraperitoneal of 9 mg Fe kg-1 b.wt. given in a volume of 10 mL kg-1 b.wt. Twenty-four h after injection, fish from each group was taken and anesthetized with 0.2 g L-1 MS-222, weighed and measured. The fish was dissected and the organs (gill, liver and kidney) were removed and prepared for histopathological analysis.
Specimen preparation for light microscopic study: The procedures for
light microscopy were modified by Humason (1972). Briefly,
the tissues were fixed in the 10% buffered formaldehyde for 24 h, dehydrate
through a graded series of ethanol and clear with xylene solutions. They were
embedded in a block using melted paraffin at the embedding station (Axel Johnson
Lab System, USA). The paraffin blocks were sectioned at 4-5 μm thickness
using a rotary microtome (HistoSTAT, Reichert, USA) and stained with Harris
hematoxylin and eosin. The tissue glass slides were examined for abnormalities
by a Nikon E600 light microscope and photographed by a Nikon DXM 1200 digital
camera (Tokyo, Japan).
Control group: The gills consisted of a row of long thin filaments,
the primary lamellae which projected from the arch like the teeth of a comb.
The surface area of each primary lamella was increased further by the formation
of regular semilunar folds across its dorsal and ventral surface, the secondary
lamellae. The primary lamellar epithelium was one or two cell layers thick.
Each secondary lamella was made up of two sheets of epithelium delimited by
many pillar cells which were contractile and separated the capillary channels.
One to two erythrocytes were usually observed within each capillary lumen. No
recognizable changes were observed in the gills of the control (G1) and guava
supplement group (G3) throughout this experiment (Fig. 1a,
||Gills of P. altus in (a) G1, showing normal arrangement
of primary (P) and secondary (s) lamellae (b) G2, showing epithelial hyperplasia
(*) and blood congestion (c) G3, no recognition change was observed (d)
G4, showing edema or epithelial lifting (arrows)
Treated groups: A variety of histological studies revealed that Fe-NTA affected gill tissue in G2. The lesions included hyperplasia, epithelial lifting or cell swelling and congestion. The lesion was started with the bending of the distal extremities of secondary lamellae, followed by a lifting of the outer layer of the lamellar epithelium, the formation of edematous spaces between the layers of epithelium which may become infiltrated with red blood cells and leukocytes. Finally, hyperplastic tissues were observed in the primary epithelial cells (Fig. 1b). Eventually, the whole epithelium sloughed off and the lamella lost its rigidity. Guava pre-obtained group (G4) showed similar but less severe alterations than those of G2 (Fig. 1d).
Control: Hepatocytes were polygonal and had a distinct central nucleus
with densely staining chromatin margins and a prominent nucleolus. Sinusoids
which were irregularly distributed between hepatocytes, were fewer in number
and were lined by endothelial cells. No recognizable changes were observed in
the hepatocytes of the control (G1) and guava supplement group (G3) throughout
this experiment (Fig. 2a, c).
Treated groups: The hepatocytes in G2 were swelling and numerous vacuolization were also observed. Blood congestion and exhibited increasing size and pyknotic nuclei were seen (Fig. 2b). However, the histological alterations were less severe in G4 (Fig. 2d).
Control: The kidney was divided into anterior and posterior portions.
The nephron of the freshwater fish was composed of a well-vascularized glomerulus,
proximal segments, distal segments and collecting duct system.
||Liver of P. altus in (a) G1, showing normal hepatocytes
and blood sinusoids (b) G2, showing severe blood congestion (arrows) (c)
G3, no recognition change was observed (d) G4, showing mild blood congestion
(arrow). CV: Central veins
||Kidney of P. altus in (a) G1, showing normal appearance
of glomerulus (G) and renal tubules (b) G2, showing renal tubule necrosis
in some area and numerous hemosiderin accumulation (arrows) (c) G3, no recognition
change was observed (d) G4, showing mild hemosiderin accumulation
No recognizable changes were observed in the glomerulus and renal tubule of
the control (G1) and guava supplement group (G3) throughout this experiment
(Fig. 3a, c).
Treated groups: There found to be some atrophy glomerulus and renal
tubular necrosis in G2. Likewise, in G4 displayed similar alterations as those
observed in G2 but they were less severe. Furthermore, there seemed to be a
few hemosiderin accumulations in some area (Fig. 3b, d).
The result of this study has shown that the aqueous extract of P. guajava
leaves were relatively very safe when taken at the tested doses. At a maximum
dose of 60 mg kg-1, no death was recorded in the experimental fish.
This safety was similar with the native fruit of Brazil, P. cattleyanum,
its leaf extract was tested in mice at concentration of 1000, 1500 and 2000
mg kg-1, failed to induce a significant increase in cell DNA damage,
in micronucleated cell frequency and in bone marrow toxicity (Costa
et al., 2008). Teixeira et al. (2003)
also evaluated the effects of the infusions of P. guajava in in vitro
and in in vivo assays on chromosomes and the cell cycle. The two different
concentrations of the infusions did not cause a statistically significant alteration
in Allium cepa L. root-tip cells, in rat cells or in cultured human lymphocytes.
Intraperitoneally injected Fe-NTA was absorbed into the portal vein through
mesothelium and passed into circulation via the liver (Umemura
et al., 1990). The present study was tested the efficiency of guava
leave supplement dietary to reduce the allied damage in vivo by Fe-NTA
toxicity. No recognition change in the histopathological analysis in the guava
supplement group (G3). Fe-NTA caused the histopathological alterations in the
gills, liver and kidney (G2 and G4). The gills were seen edema, lamellar cell
hyperplasia and epithelial lifting. There was blood congestion in sinusoids
and hepatocytes necrosis. Renal tubular swelling and also necrosis were seen.
However, fish with guava pre-obtained (G4) showed slightly alteration when compare
the only Fe-NTA treatment group (G2). These obtained results agree with our
previously study in the protective influence of ascorbic acid against the toxicity
of waterborne cadmium exposure in P. altus and lead exposure in O.
niloticus (Jiraungkoorskul et al., 2006, 2008).
The anti-inflammatory property of the aqueous leaf extract was investigated
in rats, using fresh egg albumin induced pedal edema. P. guajava aqueous
extract (50-800 mg kg-1, i.p.) produced dose dependent and significant
inhibition of fresh egg albumin-induced acute inflammation in rats (Ojewole,
2006). The hepatoprotective effect of an aqueous leaf extract of P. guajava
was studied on rat liver damage induced by carbon tetrachloride by monitoring
serum transaminase, alkaline posphatase, cholesterol, total lipids and histopathological
alterations. The leaf extract at doses of 500 mg kg-1 produced significant
hepatoprotection (Roy et al., 2006). Pretreatment
with asiatic acid (a triterpenoid extracted from P. guajava leaves and
fruit) at doses of 25, 50 or 100 mg kg-1 significantly blocked the
lipopolysaccharide and D-galactosamine-induced increases in both serum aspartate
and alanine aminotransferase levels, showing improved nuclear condensation,
ameliorated proliferation and less lipid deposition (Gao
et al., 2006). Several studies have indicated the ability of guava
to reduce several parameters associated with liver injury. Another hypothesis
of cellular damage may arise from free radicals or reactive oxygen species.
Guava leaf extracts are a potential source of natural antioxidants. These antioxidant
properties are associated with its phenolic compounds such as protocatechuic
acid, ferulic acid, quercetin and guavin B, ascorbic acid, gallic acid and caffeic
acid (Jimenez et al., 2001; Thaipong
et al., 2005). Morales et al. (1994)
showed that quercetin acts as a smooth muscle calcium antagonist. Therefore,
the protection activity of PG may be due to the presence of these compounds.
In conclusion, the results presented in this study show that the efficiency of dietary supplement of Psidium guajava leaf reducing histopathological alterations associated with exposed to ferric nitrilotriacetate uptake in fish which are cultured near heavy metal contaminated areas.
This study was funded by the Thailand Research Fund and the Commission on Higher Education: Research Grant for Mid-Career University Faculty 2008 (RMU5180001) and in part by Mahidol University International College and Faculty of Science, Mahidol University.
APHA, 2005. Standard Methods for the Examination of Water and Wastewater. 21st Edn., American Public Health Association, Washington, Dc., USA., ISBN-13: 978-0875530475.
Awai, M., M. Narasaki, Y. Yamanoi and S. Seno, 1979. Induction of diabetes in animals by parenteral administration of ferric nitrilotriacetate. A model of experimental hemochromatosis. Am. J. Pathol., 95: 663-672.
Direct Link |
Begum, S., S.I. Hassan, S.N. Ali and B.S. Siddiqui, 2004. Chemical constituents from the leaves of Psidium guajava. Nat. Prod. Res., 18: 135-140.
Costa, T.D.A., S. Vieira, S.F. Andrade and E.L. Maestro, 2008. Absence of mutagenicity effects of Psidium cattleyanum Sabine (Myrtaceae) extract on peripheral blood and bone marrow cells of mice. Genet. Mol. Res., 7: 679-686.
Direct Link |
Fracalossi, D.M., M.E. Allen, L.K. Yuyama and O.T. Oftedal, 2001. Ascorbic acid biosynthesis in Amazonian fishes. Aquaculture, 192: 321-332.
CrossRef | Direct Link |
Gao, J., J. Chen, X. Tang, L. Pan, L. Xu, L. Zhao and Q. Xu, 2006. Mechanism underlying mitochondrial protection of asiatic acid against hepatotoxicity in mice. J. Pharm. Pharmacol., 58: 227-233.
Direct Link |
Gutierrez, R.M.P., S. Mitchell and R.V. Solis, 2008. Psidium guajava: A review of its traditional uses, phytochemistry and pharmacology. J. Ethnopharmacol., 117: 1-27.
CrossRef | Direct Link |
Humason, G.L., 1972. Animal Tissue Techniques. 3rd Edn., W.H. Freeman and Co., San Francisco, ISBN: 9780716706922.
Iqbal, M., U. Giri and M. Athar, 1995. Ferric nitrilotriacetate (Fe-NTA) is a potent hepatic tumor promoter and acts through the generation of oxidative stress. Biochem. Biophy. Res. Commun., 212: 557-563.
Direct Link |
Jaiarj, P., P. Khoohaswan, Y. Wongkrajang, P. Peungvicha, P. Suriyawong, M.L.S. Saraya and O. Ruangsomboom, 1999. Anticough and antimicrobial activities of Psidium guajava leaves extract. J. Ethopharmacol., 67: 203-212.
Direct Link |
Jimenez-Escrig, A., M. Rincon, R. Pulido and F. Saura-Calixto, 2001. Guava fruit (Psidium guajava) as a new source of antioxidant dietary fiber. J. Agric. Food Chem., 49: 5489-5493.
Jiraungkoorskul, W. and S. Sahaphong, 2007. Efficacy of ascorbic acid reducing waterborne copper toxicity in butterfish (Poronotus triacanthus). J. Boil. Sci., 7: 620-625.
CrossRef | Direct Link |
Jiraungkoorskul, W., S. Sahaphong, N. Kangwanrangsan and M.H. Kim, 2006. Histopathological study: The effect of ascorbic acid on cadmium exposure in fish (Puntius altus). J. Fish. Aquatic Sci., 1: 191-199.
CrossRef | Direct Link |
Jiraungkoorskul, W., S. Sahaphong, N. Kangwanrangsan and S. Zakaria, 2008. The protective influence of ascorbic acid against the genotoxicity of waterborne lead exposure in Nile tilapia (Oreochromis niloticus). J. Fish Biol., 73: 355-366.
CrossRef | Direct Link |
Jiraungkoorskul, W., S. Sahaphong, P. Kosai and M.H. Kim, 2007. Micronucleus test: The effect of ascorbic acid on cadmium exposure in fish (Puntius altus). Res. J. Environ. Toxicol., 1: 27-36.
CrossRef | Direct Link |
Liu, M., S. Okada and T. Kawabata, 1991. Radical promoting free iron level in the serum of rats treated with ferric nitrilotriacetate, comparison with iron chelate complexes. Acta Med. Okayama, 45: 401-408.
Mercadante, A.Z., A. Steck and H. Pfande, 1999. Carotenoids from guava (Psidium guajava L.): Isolation and structure elucidation. J. Agric. Food Chem., 47: 145-151.
Morales, M.A., J. Tortoriello, M. Meckes, D. Paz and X. Lozoya, 1994. Calcium-antagonist effect of quercetin and its relation with the spasmolytic properties of Psidium guajava L. Arch. Med. Res., 25: 17-21.
Direct Link |
Ojewole, J.A., 2006. Antiinflamatory and analgesic effects of Psidium guajava Linn (Myrtaceae) leaf aqueous extract in rats and mice. Method. Find. Exp. Clin. Pharmacol., 28: 441-446.
Rattanachaikunsopon, P. and P. Phumkhachorn, 2007. Bacteriostatic effect of flavonoids isolated from leaves of Psidium guajava on fish pathogens. Fitoterapia, 78: 434-436.
Roy, C.K., J.V. Kamath and M. Asad, 2006. Hepatoprotective activity of Psidium guajava Linn. leaf extract. Ind. J. Exp. Biol., 44: 305-311.
PubMed | Direct Link |
Teixeira, R.O., M.L. Camparoto, M.S. Mantovani and V.E.P. Vicentini, 2003. Assessment of two medicinal plants, Psidium guajava L. and Achillea millefolium L. in in vivo assays. Gen. Mol. Biol., 26: 551-555.
Direct Link |
Thaipong, K., U. Boonprakob, L. Cisneros-Zevallos and D.H. Byrne, 2005. Hydrophilic and lipophilic antioxidant activities of guava fruits. South. Asian J. Trop. Med. Pub. Health, 36: 254-257.
Umemura, T.Y., K. Sai, A. Takagi, R. Hasegawa and Y. Kurakawa, 1990. Oxidative DNA damage lipid peroxidation and nephrotoxicity induced in the rat kidney after ferric nitrilotriacetate administration. Cancer Lett., 54: 95-100.
Vijayavel, K., S. Gopalakrishnan, H. Thilagan and M.P. Balasubramanian, 2006. Dietary ascorbic acid and α-tocopherol mitigates oxidative stress induced by copper in the thornfish Terapon jarbua. Sci. Total Environ., 372: 157-163.
Winkaler, E.U., T.R. Santos, J.G. Machado-Neto and C.B. Martinez, 2007. Acute lethal and sublethal effects of neem leaf extract on the neotropical freshwater fish Prochilodus lineatus. Comp. Biochem. Physiol. C: Toxicol. Pharmacol., 145: 236-244.