Lead acetate: lead acetate was purchased from El Gomhoureya For Drugs Trade & Medical Supplies (Zagazig, Egypt). Chemicals: All chemicals, unless specified otherwise, were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).

Vit. C (ascorbic acid) tablets were purchased from the local pharmacy. It is manufactured by CID Company. Each tablet contains 1g ascorbic acid.

The leaves of Ficus carica were washed with tap water and were shade dried for around one week and pulverized into coarse powder by a blender. The coarse powder was extracted with 70% aqueous EtOH by maceration at room temperature for 72 h maceration technique. The extract was subjected to filtration, and then the filtrate was concentrated in a rotary evaporator under reduced pressure 25. The dried concentrate was kept at 4° C till use.

Rats were divided into seven groups (n = 18). G₁ (Control group) got refined water. G₂ (Lead acetate group) was administered with lead acetate (20 mg/kg of body weight) using gastric tube 26. G₃ (Ficus carica group) was administered with Ficus carica leaf extract (200 mg/kg of body weight) using gastric tube 15. G₄ (Ficus extract + Lead acetate group) was administered with Ficus carica leaf extract (200 mg/kg of body weight) followed by lead acetate (20 mg/kg of body weight) after 20 minutes by using gastric tube. G₅ (Vit. C group) was administered with Vit. C (200 mg/kg of body weight) using gastric tube 27. G₆ (Vit. C + Lead acetate group) was administered with Vit. C (200 mg/kg of body weight) followed by lead acetate (20 mg/kg of body weight) after 20 minutes by using gastric tube. G₇ (Ficus carica leaf extract + Vit. C + Lead acetate group) was administered with Ficus carica leaf extract (200 mg/kg of body weight) and vitamin C (200 mg/kg of body weight) followed by lead acetate (20 mg/kg of body weight) after twenty minutes using gastric tube.

Blood samples were collected at an interval of two weeks, into clean glass tubes. The tubes were centrifuged at 3000 r.p.m to separate serum. Serum samples were transferred into Eppendorf tubes and stored at -70^oC before measuring the level of AST, ALT, ALP, TP and DB.

Serum AST and ALT activities were measured by the colorimetric method 28. Serum ALP activity was measured by the spectrophotometric method 29. Total protein was measured by a commercial kit 30. Direct bilirubin concentration was measured by the colorimetric method 31.

Liver tissues were then homogenized in 10 ml of Phosphate buffer (pH7.4). The homogenates were centrifuged at 100 000× g at 4° C for 20 min. The clear supernatant was used to measure the level of MDA, SOD, and GSH. Then parts from liver were fixed in 10 % formalin for histopathological examination. Reduced and oxidized glutathione levels were measured by HPLC-UV method 32, MDA levels were measured by HPLC-UV method 33. Superoxide dismutase (SOD) activity was measured by spectrophotometer method 34.

Finally, histopathological examination was carried out on liver tissues under different treatments 35.

Data presented as means ± SE. One-way ANOVA followed by LSD test were used to evaluate significant differences from the control and sleep deprived groups. P< 0.05 was considered to be statistically significant. Statistical processor system support (SPSS) for Windows software, release 10.0 (SPSS, Inc, Chicago, IL).

Lead acetate treatment significantly (P<0.05) increased AST, ALT and ALP levels in rats as compared with control group, after 2, 4 and 6 weeks. Both Ficus and vitamin C pre-treatment significantly (p<0.05) decreased the elevating effect of lead on AST, ALT and ALP levels after 2, 4 and 6 weeks. Combined treatment with Ficus carica and Vitamin C remarkably abolished the effect lead on AST, ALT and ALP levels (Table 1Table 2Table 3).

Table 3

Group	ALP (IU/ L) after 2 weeks	ALP (IU/ L) after 4 weeks	ALP (IU/ L) after 6 weeks
Normal control group (C)	115.52 ± 0.079 b	115.54 ± 0.162 b	115.54 ± 0.204 b
Lead acetate treated group (L)	140.21 ± 0.154 a	153.83 ± 0.112 a	162.35 ± 0.208 a
% of change of control	21.4%	33.1%	40.5%
Ficus carica treated group (Fc)	110.77 ± 0.105 a, b	113.27 ± 0.209 a, b	115.01 ± 0.069 b
% of change of control	- 4.1%	- 1.9%	- 0.46%
Ficus + Lead treated group (Fc+ L)	128.96 ± 0.249 a, b	121.69 ± 0.094 a, b	116.61 ± 0.139 a, b
% of change of control	11.6%	5.3%	0.93%
Vitamin C treated Group (Vit. C)	109.32 ± 0.179 a, b	112.72 ± 0.145 a, b	114.65 ± 0.106 b
% of change of control	- 5.4%	- 2.4%	- 0.77%
Vit. C +Lead treated group (Vit. C+ L)	131.21 ± 0.188 a, b	123.72 ± 0.111 a, b	117.83 ± 0.106 a, b
% of change of control	13.6%	7.1%	1.9%
Ficus + Vit. C +Lead treated group (Fc+ Vit. C +L)	126.8 ± 0.129 a, b	120.12 ± 0.175 a, b	115.01 ± 0.126 b
% of change of control	9.8%	3.9%	- 0.46%

Table 4 demonstrated that lead administration induced a significant reduction in TP concentration after two, four and six weeks compared to the control group. Meanwhile, administration of Ficus carica or vitamin C or Ficus carica and vitamin C caused a significant elevation in TP concentration compared to the lead-treated group.

Table 5 demonstrated that lead administration caused a significant elevation in DB level after two, four and six weeks, compared to the control group. Pre-treatment with Ficus carica or vitamin C or the combined treatment significantly minimized the elevating effect of lead on DB level when compared to the control group after two and four weeks.

Table 5

Group	DB (mg/ dl) after 2 weeks	DB (mg/dl) after 4 weeks	DB (mg/dl) after 6 weeks
Normal control group (C)	0.35± 0.025 b	0.36 ± 0.019 b	0.37 ± 0.014 b
Lead acetate treated group (L)	0.59 ± 0.013 a	0.65 ± 0.008 a	0.760 ± 0.021 a
% of change of control	71.6%	88.3%	105.4%
Ficus carica treated group (Fc)	0.27 ± 0.003 a, b	0.31 ± 0.008 a, b	0.350 ± 0.004 b
% of change of control	-21.2%	-13.3%	- 5.4%
Ficus +Lead treated group (Fc+ L)	0.48 ± 0.011 a, b	0.42 ± 0.007 a, b	0.362 ± 0.004 b
% of change of control	38.8%	16.7%	- 2.2%
Vitamin C treated Group (Vit. C)	0.27 ± 0.002 a, b	0.30 ± 0.005 a, b	0.34 ± 0.001 b
% of change of control	-22.6%	-16.6%	-8.1%
Vit. C + Lead treated group (Vit. C+ L)	0.51 ± 0.015 a, b	0.44 ± 0.008 a, b	0.371 ± 0.002 b
% of change of control	46.1%	22.2%	0.27%
Ficus +Vit C +Lead treated group (Fc+ Vit. C +L)	0.43 ± 0.005 a, b	0.38± 0.004 a, b	0.35 ± 0.002 b
% of change of control	24.9%	5.6%	- 5.4%

(a) significant difference compared to the control group (P ≤ 0.05), (b) significant difference compared to lead-treated group (P ≤ 0.05)

Table 6 showed that administration of lead caused a significant elevation in MDA level after two, four and six weeks compared to the control group. Pre-treatment with Ficus carica or vitamin C or the combined treatment significantly minimized the elevating effect of lead on MDA level when compared to the control group after two and four weeks.

Table 7 demonstrated that lead administration caused a significant decrease in GSH level after two, four and six weeks, compared to the control group. Pre-treatment with Ficus carica or vitamin C or the combined treatment significantly minimized the decreasing effect of lead on GSH level compared to the control group after two and four weeks.

Table 7

Group	GSH (μmole/g) after 2 weeks	GSH (μmole/g) after 4 weeks	GSH (μmole/g) after 6 weeks
Control group (C)	14.1±0.21 b	14.13+0.22 b	14.09+0.28 b
Lead treated group (L)	9.59±0.69 a	7.75+0.66 a	6.24+0.45 a
% of change of control	-31.9%	-45.2%	-55.7%
Ficus carica treated group (Fc)	12.7+0.24 b	13.53+0.62 b	14.05+0.59 b
% of change of control	-9.9%	-4.3%	-0.28%
Ficus + Lead treated group (Fc + L)	8.79+0.79 a	11.16+0.46 a, b	13.87+0.40 b
% of change of control	-37.7%	-21%	-1.6%
Vitamin C treated group (Vit. C)	13.28+0.57 b	13.8+0.21 b	14.01+0.52 b
% of change of control	-5.8%	-2.3	-0.66%
Vit.C + Lead treated group (Vit. C + L)	7.32+0.22 a, b	10.03+0.21 a, b	12.55+0.14 a, b
% of change of control	-48.1%	-29%	-10.9%
Ficus + Vit. C +Lead treated group (Fc+ Vit. C + L)	10.13+0.62 a	12.41+0.86 a, b	13.98+0.73 b
% of change of control	-28.2%	-12.2%	-0.78%

(a) significant difference compared to the control group (P ≤ 0.05), (b) significant difference compared to the lead-treated group (P ≤ 0.05)

Table 8 demonstrated that lead administration induced a significant reduction in SOD activity level after two, four and six weeks, compared to the control group. Pre-treatment with Ficus carica or vitamin C or the combined treatment significantly minimized the decreasing effect of lead on SOD activity compared to the control group after two and four weeks.

Histological examination of liver tissues of control group showed normal hepatocytes of hepatic lobule (Figure 1A, Figure 2A and Figure 3A). Lead acetate-treated animals exhibited parenchymatous degeneration of hepatocytes with severe necrosis and severe leucocytes infiltration throughout the experiment (Figure 1B, Figure 2B and Figure 3B). Treatment of Ficus carica extract and vit. C to control animals maintain the normal liver tissues (Figure 1C,E; Figure 2C,E and Figure 3C,E) Pre-treatment with Ficus carica extract and vit C alone or in combination remarkably provided a potential protective action against the histopatological effect of lead acetate in rat liver (Figure 1D,F,G; Figure 2D,F,G and Figure 3D,F,G).

Figure 2

Figure 3

The liver performs three principal functions which are essential to the body: detoxification of many toxins, synthesis of proteins and bile, and storage of vitamins (A, D, E, and K) and glycogen 3637. The liver is a target organ of lead toxicity 38. The present study showed that lead caused a remarkable elevation in the enzymatic activity of ALT, AST, ALP and bilirubin levels. Elevation of liver enzymes in the serum may indicate inflammation or damage to liver cells which leak higher than normal amounts of liver enzymes, into the bloodstream, which can result in higher level of liver enzymes in the blood. The elevated MDA and decreased GSH levels might indicate an increase in lipid peroxidation and oxidative stress. This effect might be interpreted that lead may induce metabolic dysfunction through inhibition of enzymatic activities and disturbance in the oxidant/antioxidant status. In accordance, a recent study indicated that oral administration of lead acetate increased the activity of blood enzymes: alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, lactate dehydrogenase and a decrease of creatinine level in rats 39. It has been suggested that lead exposure induced metabolic disorders and biochemical changes in the liver 40. Consistently, previous studies showed that lead decreased blood glutathione (GSH), glutathione peroxidase, adenosine triphosphatase, and catalase but increased oxidized GSH and intracellular calcium in rat 413.

The pretreatment with Ficus carica leaf extract or Vit. C alone or the combined treatment noticeably prevented the effect of lead on serum AST, ALT, ALP activities, and direct bilirubin and total protein concentration in the serum compared to the lead acetate treated group after two, four and six weeks. The hepatoprotective effect of both Ficus carica leaf extract and Vit. C might be attributed to their antioxidant properties. In accordance, previous studies showed that pretreatment with Ficus carica leaf extract normalized the levels of TP, ALP and the level of total serum bilirubin 4215. Moreover, Ficus carica was highly successful in attenuating lead hepatotoxicity due to its high total phenol and flavonoid contents, suggested three mechanisms for this attenuation: first: lowering the oxidative stress, second: increasing the oxidant enzymes level and third: acting as chelating agent for lead ions 4344. The observation that vitamin C caused suppression of increased ALT and AST activities induced by administration of lead acetate might be attributed to its ability to ameliorate the lipid peroxidation through the free radicals scavenging activity and restoring the liver capability 4546. Collectively, the results revealed that the pretreatment with Ficus carica leaf extract only or Vit. C only improve the hepatotoxicity while the pretreatment with the antioxidant mixture containing both Ficus carica leaf extract and Vit. C was the most effective treatment in alleviating hepatotoxicity.

In accordance with the biochemical data, the histopathological examination of the liver tissues of the animals treated with lead showed that lead acetate-induced liver hyperplasia and apoptosis, plausibly mediated by oxidative stress in Kupffer cells. Pretreatment with Ficus carica leaf extract and Vit. C provided effective protection to the liver against harmful effects induced by lead acetate.

Our data concluded that the combination of Ficus carica leaf extract and Vit. C had a critical protective action on lead acetate- induced oxidative damage in rats due to their antioxidant/anti-radical properties. The combined treatment of Ficus carica leaf extract and Vit. C offered more effective protection compared to the individual treatments.

The authors declare that they have no competing interests.

New YorkAll authors contributed equally in the study design, interpretation of the data and writing of the final manuscript.

The authors thank Dr. Omar A. Farid and Dr. Fawkya A. El-Hodairy, Physiology Department- National Organization for Drug Control and Research, for their help and great effort during practical part.

ALP: Alkaline phosphatase enzyme

ALT: Alanine aminotransferase

AST: Aspartate amino transferase

CAT: Catalase

DB: Direct bilirubin

HPLC: High-Performance Liquid Chromatography

MDA: Malondialdehyde

NODCAR: National Organization for Drug Control and Research

ROS: Reactive oxygen species

SOD: Superoxide dismutase

TP: Total protein