Abstract: 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 remains unclear.
INTRODUCTION
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 Tyrodes’s 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.
RESULTS
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 (Table 1).
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).
DISCUSSION
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 35.4%, respectively.
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.
CONCLUSION
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 testicular dysfunction.
ACKNOWLEDGMENTS
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.