Evaluation of the Effects of Citrus aurantifolia (Lime) Juice in Lead-induced Hematological and Testicular Toxicity in Rats
Background: Citrus aurantifolia (Lime) is a citrus fruit and an excellent
source of vitamin C and flavonoids which have unique antioxidant activities.
This study evaluates the protective effects of Citrus aurantifolia (Lime)
juice on lead-induced toxicity on hematological and reproductive functions and
some organ weights in the male rat. Materials and Mathods: The animals
were given Pb nitrate (4, 8 or 12 mg kg-1), or 0.1 mL Lime juice+8
mg kg-1 Pb nitrate, or vehicle daily for 28 days by oral gavage.
Results: Lead treatment caused lymphocytosis with accompanying neutropenia
and anemia. Also, lead significantly increased sperm motility, counts and viability
but did alter percentage of abnormal sperm cells (morphology). Testicular weight,
as well as liver and kidney weights were equally significantly reduced by lead
treatment. Pretreatment with lime, 1 h prior to lead (8 mg kg-1)
administration prevented hematological effects of lead. The Citrus juice also
inhibited lead-induced reductions in liver and kidney weights but not on testicular
weight. Lime pretreatment also inhibited lead-induced reduction in sperm count
but did not affect sperm motility and viability. Conclusion: Citrus
aurantifolia juice prevents lead-mediated bone marrow toxicity and may also
prevent alteration of liver and kidney functions, which may be attributed to
the antioxidants (flavonoids and vitamin C) in the plant. On the contrary, Citrus
aurantifolia juice does not protect lead-induced testicular toxicity but
may rather have potential for promoting testicular dysfunction, which mechanism
Lead is one of the oldest environmental pollutant and toxicant. Lead poisoning,
which can be traced to prehistoric time, is a major problem in most urban cities
due to its wide application in industrial activities (Bilandzic
et al., 2009). Used widely in the manufacture of batteries and ammunition
occupational exposure to the metal is high among workers in such industries
(ATSDR, 2007). Lead is also used in the manufacture of
toys, paints, X-ray apparatus (as protective shield), eye cosmetics, gasoline,
electric cables, water pipes and tanks (Stokinger, 1981;
Fischbein et al., 1992; Florea
and Busselberg, 2006). With the wide application of lead in paints and toys,
children are especially at risks of lead, even at relatively low concentrations.
The major routes of lead exposure include inhalation, gastrointestinal tract
and the skin. The inhalational and skin exposures are more significant for occupationally
exposed groups, while the latter contributes a greater proportion of those for
the general population (Florea and Busselberg, 2006).
Because of effective control measures in reducing lead use (like the stoppage
of lead in gasoline and reduction of the amount of lead in paints) by different
regulatory bodies such as the Environmental Protection Agency (EPA), Consumer
Product Safety Commission (CPSC), Centers for Disease Control (CDC) etc; acute
lead toxicity is greatly reduced. Chronic toxicity on the other hand, is much
more common and occurs at blood lead levels of 40-60 μg dL-1,
which in severe cases, is characterized by persistent vomiting, anemia, encephalopathy,
lethargy, delirium, convulsions and coma (CDCP/NCEH, 2005).
Several systems in the body could become targets in lead intoxication. The
main targets include the hematopoietic (Anetor et al.,
2002; Flora et al., 2006), nervous (White
et al., 2007) and cardiovascular systems (Navas-Acien
et al., 2007). Lead also affects renal (Ekong
et al., 2006) and reproductive functions (ATSDR,
2007; Makhlouf et al., 2008; Zhu
et al., 2010). Existing data suggest that lead exposure results in
oxidative stress and this has been reported to be the major mechanism of lead
toxicity in most biological systems (ATSDR, 2005; Flora
et al., 2012).
Citrus aurantifolia (Lime) is a citrus fruit and an excellent source
of vitamin C and flavonoids which have unique antioxidant activities (Patil
et al., 2009; Shrestha et al., 2012;
Karoui and Marzouk, 2013). Citrus aurantifolia,
which belongs to the Rutaceae family, is believed to reduce oxidative stress
by inhibiting cellular lipid peroxidation and increasing cellular antioxidant
systems (Shrestha et al., 2012; Karoui
and Marzouk, 2013). Gharagozloo and Ghaderi, 2001;
Patil et al. (2009) have also reported that
Citrus aurantifolia (Lime) protect against proliferation of cancer cells
but there is limited data on its protective effects in other metal induced toxicities.
Since Citrus aurantifolia juice contains potent antioxidants and such
antioxidants have been demonstrated to prevent metal-induced toxicities (Gurer
et al., 1998; Obianime et al., 2010;
Sajitha et al., 2010; Ayinde
et al., 2012), it is logical to hypothesize that Citrus aurantifolia
juice will protect lead-induced organ toxicities in the rat. The present study
aims to evaluate the protective effects of Citrus aurantifolia (Lime)
juice on lead-induced toxicity on hematological and reproductive functions and
some organs in male rats.
MATERIALS AND METHODS
Lead nitrate (Burgoyne Burgoynes and Co, India) was obtained from the Department
of Pharmacology, University of Port Harcourt.
Plant material: Fresh fruits of Citrus aurantifolia used in this research were obtained from an orchad located at the University of Port Harcourt, Nigeria. The fruits were harvested from three trees and juice was expressed out, filtered with muslin and used fresh for the experiments.
Animals: Adult male Wistar albino rats, weighing between 180-230 g were
obtained from the Animal House of the Department of Pharmacology, University
of Port Harcourt and used for the study. The animals were maintained in a ventilated
room at a room temperature of 28.0±2°C and natural lighting condition.
The animals were fed with standard rodents chow and water was given ad libitum.
The animals were handled in accordance with the Guide for Care and Use of Laboratory
Animals (CCAC, 2009) and the experimental protocol was
approved by the Committee for Ethics in Animal Experimentation of the University
of Port Harcourt.
Experimental design: The animals were randomly distributed into five
groups (n = 6) and administered with different concentrations of Pb as nitrate
daily for 28 days by oral gavage. Group I was given vehicle (distilled water)
and was used as the control. Group II was given 4 mg kg-1 (≈27
ppm) Pb. Group III received 8 mg kg-1 (≈54 ppm) Pb. Group IV
received 12 mg kg-1 (≈80 ppm) Pb. Group V was given Citrus
aurantifolia juice (0.1 mL) for 1 h before administering 8 mg kg-1
(≈54 ppm) Pb. At the end of the drug administrations, the rats were sacrificed
under deep diethylether anesthesia. Blood was collected into EDTA bottles and
hematological parameters were measured. The testes were excised out, weighed
and sperm was collected from the epididymis to analyze sperm count, motility,
morphology and viability. Animal livers and kidneys were also removed and weighed.
Hematological analysis: Whole blood collected into EDTA bottle was assayed for Packed Cell Volume (PCV), White Blood Cell (WBC) counts and WBC differentials lymphocyte and neutrophil counts using an auto analyzer.
Sperm analysis: The method of Amelar et al.
(1973) was used in collecting sperm cells from the epididymis. Briefly,
the testis was excised and the caudal epididymis was carefully isolated and
placed in a Petri dish containing 3 mL of NaHCO3 buffered Tyrodess
Lactate solution. Several (1 mm) incisions were made on it and sperm was gently
drawn into a plastic transfer pipette and transferred into 5 mL test tubes and
vigorously shaken for homogeneity and dispersal of sperm cells. Sperm was then
analyzed to determine sperm motility, sperm count, percentage of abnormal sperm
cells (sperm morphology) and percentage of viable sperm cells (sperm viability)
following standard procedures (WHO, 1999).
Statistical analysis: Data were expressed as Mean±SEM. Statistical differences between the groups were evaluated by one way ANOVA, followed by Dunnets comparison test to compare between treated and control groups. Differences yielding p<0.05 were considered statistically significant. Statistical analyses of data were performed using GraphPad Prism 5 software.
Hematological indices: Hematocrit or Packed Cell Volume (PCV) values obtained
in Groups III and IV were significantly lower than control rats (Table
1). White Blood Cell (WBC) counts of only lead treated rats were higher
compared to control but only that of group IV was significant, p<0.05 (Table
1). Similarly, lymphocyte counts of only lead treated rats were higher compared
to control but only that of group IV was significant, p<0.05 (Table
1). Neutrophil counts of only lead treated rats were decreased but group
IV value was also significant, p<0.05 when compared to the control (Table
1). The blood counts of WBC and its differentials in Citrus aurantifolia
juice pretreated rats (Group V) were not all significantly different from controls
Sperm parameters: Mean epididymal sperm count, motility and viability obtained in Pb-treated rats (Groups II, III and IV) were dose-dependently decreased. However, only the sperm motility and viability values in Groups III and IV (8, 12 mg kg-1 Pb-treated) rats were significant (Group III, p<0.01,p<0.05; Group IV, p<0.001,p<0.01), compared to Group I (control) rats, (Table 2). These Pb-induced levels corresponded to 45.5 and 56.8% decreases, respectively in sperm motility and 37.3 and 43.7% decreases, respectively in sperm viability. Also, only the sperm count in Group IV (12 mg kg-1 Pb-treated) rats was significant, p<0.05 (Table 2), which was equivalent to 22.6% decrease, compared to the control. Percentages of abnormal sperm cells (sperm morphology) in Pb-treated rats (Groups II, III and IV) were not significantly different from control rats (Table 2). Furthermore, while sperm count obtained in Citrus aurantifolia pretreated rats (Group V) was not significantly altered, sperm motility and viability were decreased, p<0.001,p<0.01 when compared to control rats (Table 2). The respective level of reductions in motility and viability were 61.4 and 47.5%. There was no significant difference in the percentage of abnormal sperm cells (sperm morphology) between (Group V) and control rats (Table 2).
Organ weights: Weights of testis, liver and kidney in Pb-treated rats
(Groups II, III and IV) were significantly decreased in dose-dependent manners,
compared to Group I (control) rats (Table 3).
Weight of testis of Citrus aurantifolia-pretreated rats (Group V) were
also significantly lower than control rats but there were no changes in liver
and kidney weights between Citrus aurantifolia-pretreated (Group V) and
control rats (Table 3).
Lead is a widespread environmental pollutant associated with toxicity of
many body functions, including the hematopoietic and reproductive systems in
humans and experimental animals (Chandra et al.,
1981; Anetor et al., 2002; Bellinger,
2005; Ekong et al., 2006; ATSDR,
2007). Low Packed Cell Volume (PCV) or hematocrit, which results in anemia,
is a common sign of lead toxicity (Jacob et al.,
2000; Anetor et al., 2002). This supports
the observation of the reduction of PCV by lead in this study. Chronic exposure
of high levels of lead (2,000 ppm) has been reported to induce significant immunosuppression
in rats (Ercal et al., 2000). The results of
this study however, indicate that low levels of lead exposure would cause elevation
in white blood cell (WBC) levels with proliferation of lymphocytes and reduction
of neutrophil levels, indicative of bone marrow toxicity. This observation suggests
that responses of immunological cells to lead may depend on its concentration.
The lead-induced lymphocytosis with accompanying neutropenia and anemia has
a high implication for the development of lymphoproliferative neoplasms (Dhodapkar
et al., 1994; George, 2012).
Testicular function is assessed in part by analysis of spermatic indices including
sperm count, motility, viability and morphology (Zinaman
et al., 2000; Eliasson, 2003). Measurements
of these parameters in the spermatozoa give an indication of the quality and
functionality of the sperm. As normal sperm motility and count are vital for
male fecundity (Zinaman et al., 2000), reductions
in the number and motility of sperms by lead in this study indicates that the
metal could alter normal testicular function. Additionally, the percentages
of viable sperms (i.e., spermatozoa with intact cell membrane) and morphologically
abnormal sperms are critical indicators of testicular function (Menkveld
et al., 2011). Lead-induced decrease in viability and increase in
morphologically abnormal sperm cells observed in this study is strongly suggestive
of testicular dysfunction. Organ weights are indices often employed in toxicological
evaluations (Michael et al., 2007). Reduction
in testis and other organ weights in lead treated rats in this study demonstrate
lead toxicity. This observation also suggests that lead may cause atrophy of
the testis, liver and kidney and ultimately affect their physiological functions.
Earlier studies have reported that Citrus aurantifolia extracts protect
against lead induced bone marrow toxicity (Gharagozloo
and Ghaderi, 2001; Hosseinimehr et al., 2003;
Patil et al., 2009). However, lead is deleterious
to several biological systems and the protective potential of Citrus aurantifolia
on other organ functions, including reproductive function has not been studied.
It is important to note that the fruit juice of Citrus aurantifolia is
more commonly used than most of the other parts of the plant. It also has a
higher vitamin C and flavonoid contents than other parts of the plant (Karoui
and Marzouk, 2013). Unfortunately, Citrus aurantifolia fruit juice
has not been assessed for the prevention or amelioration of heavy metal induced
toxicity, even though similar antioxidant compounds have been shown to exhibit
beneficial properties in this regard (Gurer et al.,
1998; Obianime et al., 2010; Sajitha
et al., 2010; Ayinde et al., 2012).
In the present study, Citrus aurantifolia fruit juice prevented the hematological
effects of lead (anemia, lymphocytosis and neutropenia), just as it inhibited
the lead-induced reduction of sperm count. However, the low percentages of sperm
motility and viability observed in Citrus aurantifolia juice pretreated
rats indicate that the effects of lead on these sperm indices were unchallenged
by Citrus aurantifolia juice. Additionally, no effect observation on
liver and kidney weights in Citrus aurantifolia juice pretreated rats
clearly shows that lead-induced atrophy of these organs was inhibited. Interestingly,
Citrus aurantifolia juice failed to prevent testicular atrophy caused
by lead, similar to its effects on sperm motility and viability. When the level
of alterations in these parameters were analyzed between only lead treated and
Citrus aurantifolia juice pretreated rats, it was observed that sperm
motility was reduced by 56.8 and 61.4%, respectively; sperm viability was reduced
by 43.7 and 47.5%, respectively and weight of testis was reduced by 62.2 and
Citrus aurantifolia contains antioxidants that may be responsible for
the amelioration of lead-mediated toxicity of bone marrow, liver and kidney.
However, the findings of the present study provide evidence that Citrus aurantifolia
juice may not protect lead-induced testicular damage. With higher levels of
alterations in some of the spermatic indices by Citrus aurantifolia juice
pretreatment, compared to only lead treatment, Citrus aurantifolia juice
may independently cause adverse effects on testicular function and indeed have
antifertility potential in the male. Besides antioxidants, Citrus aurantifolia
juice also contains high amounts of organic acids like citric acid, coumaric
and ferulic acids (Patil et al., 2009; Shrestha
et al., 2012). As testicular milieu is highly sensitive to most chemicals,
the testicular effects of Citrus aurantifolia juice may be attributed
partly to the acid constituents of the plant.
The results of this study show that Citrus aurantifolia juice may
protect lead mediated bone marrow toxicity and alteration of liver and kidney
functions. On the contrary, Citrus aurantifolia juice does not protect
lead-induced testicular toxicity but may rather have potential for promoting
Authors are grateful to Mr. M.D. Gwotmot, Chief Lab Scientist of the Department
of Human Physiology and the laboratory staff of the Department of Pharmacology,
University of Port Harcourt, Nigeria for their technical assistance.
Amelar, R.D., L. Dubin and C. Schoenfeld, 1973.
Semen analysis: An office technique. Urology, 2: 606-611.Direct Link |
Anetor, J.I., O.O. Babalola, F.A.A. Adeniyi and T.S Akingbola, 2002.
Observations on the heamopoietic system in tropical lead poisoning. Nig. J. Physiol. Sci., 17: 9-15.
Toxicological Profile for Lead. U. S Department of Health and Human Services, Atlanta, Georgia
Toxicological Profile for Lead. Department of Health and Human Services, ATSDR, Atlanta, USA
Ayinde, O.C., S. Ogunnowo and R.A. Ogedegbe, 2012.
Influence of vitamin C and vitamin E on testicular zinc content and testicular toxicity in lead exposed albino rats. BMC Pharmacol. Toxicol., Vol. 13.CrossRef | Direct Link |
Bellinger, D.C., 2005.
Teratogen update: Lead and pregnancy birth defects. Res. A. Clin. Mol. Teratol., 73: 409-420.Direct Link |
Bilandzic, N., M. Sedak, D. Vrataric, T. Peric and B. Simic, 2009.
Lead and cadmium in red deer and wild boar from different hunting grounds in Croatia. Sci. Total Environ., 407: 4243-4247.CrossRef | PubMed |
The Care and Use of Farm Animals in Research, Teaching and Testing. CCAC., Ottawa, ON., pp: 12-15Direct Link |
Third National Report on Human Exposure to Environmental Chemicals. CDC, National Center for Environmental Health, Atlanta, GA., ISBN: 0309071402, Pages: 12
Chandra, A.V., M.M. Ali, D.K. Saxena and R.C. Murthy, 1981.
Behavioral and neurochemical changes in rats simultaneously exposed to manganese and lead. Arch. Toxicol., 49: 49-56.CrossRef | Direct Link |
Dhodapkar, M.V., C.Y. Li, J.A. Lust, A. Tefferi and R.L. Phyliky, 1994.
Clinical spectrum of clonal proliferations of T-large granular lymphocytes: A T-cell clonopathy of undetermined significance? Blood, 84: 1620-1627.PubMed |
Ekong, E.B., B.G. Jaar and V.M. Weaver, 2006.
Lead-related nephrotoxicity: A review of the epidemiologic evidence. Kidney Int., 70: 2074-2084.CrossRef | Direct Link |
Eliasson, R., 2003.
Basic Semen Analysis. In: Current Topics in Andrology, Matson, P. (Ed.). Ladybrook Publishing, Perth, WA, pp: 35-89
Ercal, N., R. Neal, P. Treeratphan, P.M. Lutz, T.C. Hammond, P.A. Dennery and D.R. Spitz, 2000.
A role for oxidative stress in suppressing serum immunoglobulin levels in lead exposed Fisher 344 rats. Arch. Environ. Contam. Toxicol., 39: 251-256.PubMed | Direct Link |
Fischbein, A., J. Wallace, S. Sassa, A. Kappas, G. Butts, A. Rohl and B. Kaul, 1992.
Lead poisoning from art restoration and pottery work: Unusual exposure source and household risk. J. Environ. Pathol. Toxicol. Oncol., 11: 7-11.CrossRef | PubMed |
Flora, G., D. Gupta and A. Tiwari, 2012.
Toxicity of lead: A review with recent updates. Interdiscip. Toxicol., 5: 47-58.CrossRef | PubMed | Direct Link |
Flora, S.J.S., G. Flora and G. Saxena, 2006.
Environmental Occurrence, Health Effects and Management of Lead Poisoning. In: Lead Chemistry, Analytical Aspects, Environmental Impacts and Health Effects, Cascas, S.B. and J. Sordo (Eds.). Elsevier Publication, Netherlands, pp: 158-228
Florea, A.M. and D. Busselberg, 2006.
Occurrence, use and potential toxic effects of metals and metal compounds. Biometals, 19: 419-427.CrossRef |
George, T.I., 2012.
Malignant or benign leukocytosis. Hematol. Am. Soc. Hematol. Educ. Program., 2012: 475-484.PubMed |
Gharagozloo, M. and A. Ghaderi, 2001.
Immunomodulatory effect of concentrated lime juice extract on activated human mononuclear cells. J. Ethnopharmacol., 77: 85-90.CrossRef | PubMed |
Gurer, H., H. Ozgunes, R. Neal, D.R. Spitz and N. Ercal, 1998.
Antioxidant effects of N
-acetylcysteine and succimer in red blood cells from lead-exposed rats. Toxicology, 128: 181-189.CrossRef | Direct Link |
Hosseinimehr, S.J., H. Tavakoli, G. Pourheidari, A. Sobhani and A. Shafiee, 2003.
Radioprotective Effects of Citrus Extract Against Gamma-Irradiation in Mouse Bone Marrow Cells. J. Radiat. Res., 44: 237-241.PubMed | Direct Link |
Jacob, B., B. Ritz, J. Heinrich, B. Hoelscher and H.E. Wichmann, 2000.
The effect of low-level blood lead on hematologic parameters in children. Environ. Res., 92: 150-159.CrossRef | PubMed |
Karoui, I.J. and B. Marzouk, 2013.
Characterization of bioactive compounds in tunisian bitter orange (Citrus aurantium
L.) peel and juice and determination of their antioxidant activities. BioMed. Res. Int.,CrossRef | Direct Link |
Makhlouf, M.M.M., H.M.S. Eldien, D.A.M. Zagloul, E.A.B. Dief and N.G. Abd ElHaliem, 2008.
The effect of lead acetate on testicular structure and protective effect of vitamin e in adult albino rat. Egypt. J. Histol., 31: 406-418.Direct Link |
Menkveld, R., C.A.G. Holleboom and J.P.T. Rhemrev, 2011.
Measurement and significance of sperm morphology. Asian J. Androl., 13: 59-68.CrossRef | PubMed |
Michael, B., B. Yano, R.S. Sellers, R. Perry and D. Morton et al
Evaluation of organ weights for rodent and non-rodent toxicity studies: A review of regulatory guidelines and a survey of current practices. Toxicol. Pathol., 35: 742-750.CrossRef | PubMed | Direct Link |
Navas-Acien, A., E. Guallar, E.K. Silbergeld and S.J. Rothenberg, 2007.
Lead exposure and cardiovascular disease: A systematic review. Environ. Health Perspect., 115: 472-482.CrossRef | PubMed |
Obianime, A.W., N.J. Ahiwe and J.S. Aprioku, 2010.
Effects of vitamins C and E pretreatments on cadmium-induced serum levels of some biochemical and hormonal parameters in the female guinea pig. Afr. J. Biotechnol., 9: 6582-6587.Direct Link |
Patil, J.R., K.N. Chidambara Murthy, G.K. Jayaprakasha, M.B. Chetti and B.S. Patil, 2009.
Bioactive compounds from Mexican lime (Citrus aurantifolia
) juice induce apoptosis in human pancreatic cells. J. Agric. Food Chem., 57: 10933-10942.CrossRef | PubMed |
Sajitha, G.R., R. Jose, A. Andrews, K.G. Ajantha, P. Augustine and K.T. Augusti, 2010.
Garlic oil and vitamin E prevent the adverse effects of lead acetate and ethanol separately as well as in combination in the drinking water of rats. Indian J. Clin. Biochem., 25: 280-288.CrossRef |
Shrestha, R.L., D.D. Dhakal, D.M. Gautum, K.P. Paudyal and S. Shrestha, 2012.
Variation of physiochemical components of acid lime (Citrus aurantifolia
swingle) fruits at different sides of the tree in Nepal. Am. J. Plant Sci., 3: 1688-1692.Direct Link |
Stokinger, H.E., 1981.
The Metals. In: Patty's Industrial Hygiene and Toxicology, Clayton, G.D. and F.E. Clayton (Eds.). 3rd Edn., John Wiley and Sons, New York, pp: 1687-724
White, L.D., D.A. Cory-Slechta, M.E. Gilbert, E. Tiffany-Castiglioni and N.H. Zawia et al
New and evolving concepts in the neurotoxicology of lead. Toxicol. Applied Pharmacol., 225: 1-27.CrossRef | PubMed |
WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th Edn., Cambridge University Press, New York, USA., ISBN-13: 9780521645997, Pages: 128
Zhu, M., E.F. Fitzgerald, K.H. Gelberg, S. Lin and C.M. Druschel, 2010.
Maternal low-level lead exposure and fetal growth. Environ. Health Perspect., 118: 1471-1475.PubMed |
Zinaman, M.J., C.C. Brown, S.G. Selevan and E.D. Clegg, 2000.
Semen quality and human fertility: A prospective study with healthy couples. J. Androl., 21: 145-153.Direct Link |