Amelioration of lead toxicity on rat liver with Vitamin C and silymarin supplements

Toxicology 206 (2005) 1–15

Review

Amelioration of lead toxicity on rat liver with Vitamin C and silymarin supplements M.G. Shalana,∗, M.S. Mostafab, M.M. Hassounab, S.E. Hassab El-Nabic, A. El-Refaied a

Biological and Geological Sciences Department, Al-Arish Faculty of Education, Suez Canal University, Center of Town, Al-Arish, North Sinai 02, Egypt b Clinical Pathology Department, National Liver Institute, Menoufiya University, Egypt c Zoology Department, Faculty of Science, Menoufiya University, Egypt d Pathology Department, National Liver Institute, Minoufiya University, Egypt Received 9 June 2004; received in revised form 9 July 2004; accepted 12 July 2004 Available online 25 August 2004

Abstract The aim of the present study was to investigate the impact of the combined administration of Vitamin C and silymarin on lead toxicity. Male albino rats were subdivided into three groups: the first was a control group, the second received lead acetate in diet as 500 mg/kg diet daily, the third received the same lead acetate dose and supplemented with Vitamin C (1 mg/100 g body weight) and silymarin (1 mg/100 g body weight) by gastric tube three times per week. Blood samples were taken after 2, 4 and 6 weeks of treatment. Significant lead-induced elevations in serum ALT, AST, GGT and ALP activities were observed after different periods of treatment. However, serum LDLc was decreased. The intensities of RNA and apoptotic fragments of DNA were measured as optical density by Gel-pro program. Lead acetate decreased the intensity of DNA at 6 weeks and induced apoptotic DNA fragments reversibly with time. After 2 weeks of lead administration dilation and congestion of terminal hepatic veins and portal vein branches were observed. Lead also induced hepatocyte proliferation without any localized distribution among zones 1–3. Portal inflammatory infiltrate with disruption of the limiting plates (interface hepatitis), steatosis, apoptosis and mild fibrosis were detected especially by sixth week of lead administration. Combined treatment of lead–exposed animals with Vitamin C and silymarin showed marked improvement of the biochemical, molecular and histopathological findings. These experimental results strongly indicate the protective effect of Vitamin C and silymarin against toxic effects of lead on liver tissue. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Lead; Vitamin C; Silymarin; Liver; Serum

∗

Corresponding author. Tel.: +20 482 2275 70; fax: +20 683 500 65. E-mail address: [email protected] (M.G. Shalan).

0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.07.006

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Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

2.

Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Duration time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Collection of serum samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Preparation of tissues for microscopical and gel examinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Histopathological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Gel preparation and electrophoresis of lysate tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Nucleic acids extraction and molecular assessment for apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Apoptosis analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10. Biochemical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 3 3 3 3 3 3 4 4 4 4

3.

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Histopathological findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 4 4 6

4.

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Lead and its compounds play a significant role in modern industry; a wide variety of population were at risk of occupational exposure and lead is suspected to be a human carcinogen (Fracasso et al., 2002). Lead has many undesired effects, including neurological (Royce et al., 1990), behavioral (Shafiq-ur-Rehman, 1991), respiratory (Hillam and Ozkan, 1986), visual (Winneke et al., 1988), growth retardation (Shukla et al., 1991), hematological (Falke and Xwennis, 1990), immunological (Sroczynski et al., 1987), renal (Vyskocil et al., 1989, 1991), hepatic (Honchel et al., 1991; Hao and Tian, 2002) and reproductive disfunctions (Marchlewicz et al., 1993; Winder, 1993). The biochemical and molecular mechanisms of lead toxicity are poorly understood, but emerging data suggest that some of the effects of lead may be due to its interference with calcium in the activation of protein kinase C (PKC) and or through production of reactive oxygen species (ROS). Also, it was reported that lead increased the level of lipid peroxidation (Upasani et al., 2001). Low dose lead acetate administration was found to induce slight decrease in hepatic RNA content (Stone and Fox, 1984), however, high dose administration of lead acetate was recorded to increase hepatic RNA content (Dhar and Banerjee, 1983). Kristal-Boneh

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et al. (1999) showed that subjects exposed to lead had higher mean levels of total cholesterol and HDL cholesterol, however, Skoczynska and Smolik (1994) showed that rats poisoned with lead acetate displayed lower total and HDL cholesterol levels in comparison to controls, but this was associated with increase of free cholesterol concentration and hypertriglyceridemia. On the other hand, Antonowicz and Andrzejak (1996) found no significant disturbances in lipid metabolism in workers chronically exposed to heavy metals. Skoczynska et al. (1993) showed that in rats poisoned with small doses of lead, a decrease in plasma cholesterol level and HDL-cholesterol fraction were observed. Vitamin C (ascorbic acid), as a chelating agent is reported in treatment of lead toxicity (Llobet et al., 1990). It reduces the possibility of lead interacting with critical biomolecules and factors inducing oxidative damage (Hsu and Guo, 2002). It acts as a free radical scavenger (Kleszczewska, 2001) and reduces the level of lipid peroxidation (Upasani et al., 2001). On the other hand, Simon and Hudes (1999) suggested that high serum levels of ascorbic acid are independently associated with a decreased prevalence of elevated blood lead levels, therefore its intake may have public health implications for control of lead toxicity.

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Silymarin is a standardized mixture of antioxidant flavonolignans (silybin and silibinin) extracted from the medicinal plant Silybum marianum. It is a free radical scavenger and a membrane stabilizer that prevents lipoperoxidation and its associated cell damage in some experimental models (Soto et al., 1998). Silymarin was proved to have a protective effect against experimental hepatotoxicity by regulating the actions of the ultrastructures of the liver cells, and improving the performance of hepatic enzymes and bile production (Hagymasi et al., 2002; Lucena et al., 2002). Hence lead is removed actively, presumably via hepatic bile (P’an and Kennedy, 1989), silymarin can be used effectively for lead elimination. Silymarin possess anti-inflammatory activities mediated by alteration of kupffer cell functions (Dehmlo et al., 1996). It has been shown to have immunomodulatory effects by decreasing the number and the cytotoxic activity of CD8 lymphocytes (Leuschner et al., 1990). In addition, silymarin has antifibrotic activities through effects on transforming growth factor ␤ and matrix gene expression by hepatic stellate cell (Fuchs et al., 1995). It was reported that treatment of lead toxicity with Vitamin C alone resulted in reversal of oxidative stress without a significant decline in tissue lead burden (Patra et al., 2001). On the other hand, supplementation with antioxidant mixture containing both silymarin and Vitamin C helped in improving lipid peroxidation, hypoxia and liver functions (Zou et al., 2001; Schreiber and Trojan, 1998; Halim et al., 1997). Thus the present study investigated the protective effect of combined supplementation with Vitamin C and silymarin, against the pollution of diet with lead.

2.2. Experimental design

2. Material and methods

2.6. Histopathological methods

2.1. Animals

Five-micron thick histological sections were prepared and stained with hematoxyline and eosin, Masson’s trichrome stain and sirius red stains for collagen and Perl’s stain for iron. Microscopic analysis of the specimens was done blindly. Histopathological assessment of the inflammatory activity and stage of fibrosis was performed according to the modified Knodell’s sconing system (Ishak et al., 1995).

Forty-five (45) male albino rats (Rattus norvigicus), purchased from the Egyptian Organization for Biological and Vaccine Production, A.R.E., 8 weeks old, weighing about 100 ± 20 g were used as experimental animals. Throughout the present investigation, animals were housed in-groups in plastic cages.

Animals were segregated into three groups: 1. Normal controls. 2. Lead intoxicated group: received (500 mg lead acetate/kg diet) daily and water ad libitum. 3. Vitamin C and silymarin supplemented group: received (500 mg lead acetate/kg diet) daily and supplemented with 1 mg Vitamin C/100 g body weight and 1 mg silymarin/100 g body weight by gastric tube three times per week. 2.3. Duration time Animals of different groups were anaesthetized and rapidly dissected after 2, 4 and 6 weeks of treatment. 2.4. Collection of serum samples Blood samples were collected from the abdominal vein in glass centrifuge tubes, then centrifuged for 15 min at 1000 × g. Sera were separated and stored at −30 ◦ C in deep freezer till further biochemical measurements. 2.5. Preparation of tissues for microscopical and gel examinations After animal dissection liver was removed, blotted on filter paper and weighted. Representative specimens were placed in Bouin’s solution and processed to paraffin for histological section and staining. Portions of 10 mg were taken immediately for gel examinations and the remaining portions were stored at −30 ◦ C.

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2.7. Gel preparation and electrophoresis of lysate tissue Gels were prepared with 1.8% electrophoretic grade agarose (BRL). The agarose was boiled in Tris–borate EDTA buffer (1× TBE buffer; 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). The 0.5 ␮g/ml ethidium bromide was added to gel at 40 ◦ C. Gels were poured and allowed to solidify at room temperature for 1 h before samples were loaded. The 10 mg hepatic tissue was squeezed and lysed in 200 ␮l lysing buffer (50 mM NaCl, 1 mM Na2 EDTA, 0.5% SDS, pH 8.3) for at least 30 min. For electrophoretic pattern of nucleic acids of tissue lysate, 20 ␮l of lysate hepatic cells was loaded in well, 5 ␮l 6× loading buffer was added on the lysing tissue. Electrophoresis was performed for 2 h at 50 V in gel buffer (1× TBE buffer). Gel was photographed using a Polaroid camera while the DNA and RNA were visualized using a 312 nm UV transilluminator. 2.8. Nucleic acids extraction and molecular assessment for apoptosis Nucleic acids extraction based on salting out extraction method (Aljanabi and Martinez, 1997), whereas protein was precipitated by saturated solution of NaCl (5 M). The 10 mg hepatic tissue was squeezed in eppendorf tube and was lysed by 600 ␮l lysing buffer (50 mM NaCl, 1 mM Na2 EDTA, 0.5% SDS, pH 8.3) and was shacked gently. The mixture was kept overnight at 37 ◦ C. For protein precipitation, an amount of 200 ␮l of saturated NaCl was added to the samples and then shacked gently and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to new eppendorf tube and the DNA was precipitated by 700 ␮l cold iso-propanol. The mixture was inverted several times till fine fibers of nucleic acids appear and centrifuged for 10 min at 12,000 rpm. The supernatant is removed. For washing, an amount of 500 ␮l 70% ethyl alcohol was added and centrifuged for 8 min at 12,000 rpm. The supernatant was decanted or tipped and the tubes blotted on Whatman paper or clean tissue for 15 min. For apoptosis, once the tube was seen to be dry, the pellet was resuspended in 50 ␮l or appropriate volume of TE buffer (10 mM Tris, 1 mM EDTA, pH 8), supplemented with 5% glycerol for 30 min. To get red of RNA, an appropriate volume of RNase was added and

incubated at 37 ◦ C for 1 h. The resuspended DNA with 6× loading buffer was loaded directly on gel. So, the fine apoptotic bands could be detected even in control. 2.9. Apoptosis analysis For apoptosis, the extracted DNA was gently resuspended with TE buffer supplemented with 5% glycerol, gently pipetting, and the RNA digested with RNase for 1 h, then the samples were mixed with 6× loading buffer, and loaded directly on the gel (Hassab El-Nabi, 2004). The remained DNA was kept at −20 ◦ C for another loading. Apoptotic bands appear and located at 180 bp and it’s multiply. The intensity of apoptotic bands could be measured by Gel-Pro program. 2.10. Biochemical analysis Serum total lipids were determined colorimetrically with sulfophosphovanillinic mixture (Knight et al., 1972). Serum cholesterol, triglycerides, LDLc, HDLc, total protein, glucose, ALT, AST, GGT and ALP levels were determined automatically using Integra 800 autoanalyzer, Liver Institute, Menoufiya University. Lead was determined in serum samples by atomic absorption spectrophotometry using G.B.C. 902, double beam atomic absorption spectrophotometer (A.D.S. instrument), Faculty of Agriculture, Menoufiya University.

3. Results 3.1. Biochemistry Chronic lead ingestion resulted in a significant increase in serum ALT, AST, GGT and ALP levels after 2, 4 and 6 weeks of treatment (Table 1). Blood lead level was increased on lead acetate diet ingestion (Table 2). A significant decrease in low-density lipoprotein cholesterol concentration was reported after lead intake. However, supplementation with Vitamin C and silymarin normalized these effects. 3.2. Molecular biology Fig. 1 shows the optical density of basal and apoptotic fragments of DNA at 180, 360 and 540 bp of liver cells of different groups. The basal DNA decreased in

Table 1 Changes in serum ALT, AST, GGT and ALP activities of male rats in different animal groups 2-Week treatment

ALT (U/L) AST (U/L) GGT (U/L) ALP (U/L)

a

Control

Lead

Difference (%)

L + VC + S

41.66 ± 5.37 187 ± 22.8 21.25 ± 2.4 170.66 ± 12.5

50.4 ± 5.15 217.6 ± 20.83 25.8 ± 3.7 204.4 ± 25.8

20.97∗ 16.36∗ 21.41∗ 19.77∗

43.5 ± 5.31 200.33 ± 21.12 22.75 ± 2.04 171.2 ± 18.3

Control

Lead

L + VC + S

41.50 ± 4.51 162.5 ± 16.5 19.31 ± 2.05 176 ± 17.59

52.87 ± 5.96 192.5 ± 20.4 24.1 ± 2.23 204.5 ± 20.5

Difference (%) 27.40∗∗ 18.46∗ 24.81∗∗ 16.19∗

Difference (%) 4.42 7.13 7.06 0.32

4-Week treatment Control

Lead

41.33 ± 4.51 177 ± 13.52 20.37 ± 1.50 174.5 ± 17.67

51.4 ± 5.61 206.4 ± 22.37 25.1 ± 3.77 214 ± 24.09

Difference (%) 24.36∗∗ 16.61∗ 23.22∗∗ 22.63∗

L + VC + S 45.4 ± 4.39 178.6 ± 19.7 21.75 ± 1.91 178 ± 18.5

Difference (%) 9.85 0.9 6.77 2.01

6-Week treatment

46.62 ± 5.11 175.5 ± 32.78 20.30 ± 2.13 178.1 ± 17.9

Number of rats = 5; values represents mean ± standard deviation. a Percentage difference of controls. ∗ Significant at P < 0.05. ∗∗ Highly significant at P < 0.01.

Table 2 Effect of lead acetate on serum metabolites, blood lead level and the prophylactic action of Vitamin C and silymarin supplementation N=5

Glucose (mg/dl)

Control (2 weeks) Lead (2 weeks) Percentagea (2 weeks) VC + S + L (2 weeks) Percentage (2 weeks) Control (4 weeks) Lead (4 weeks) Percentage (4 weeks) VC + S + L (4 weeks) Percentage (4 weeks) Control (6 weeks) Lead (6 weeks) Percentage (6 weeks) VC + S + L (6 weeks) Percentage (6 weeks)

111.6 ± 12.40 127.1 ± 13.7 13.88 125.5 ± 11.44 12.45 109.6 ± 11.14 125.4 ± 12.61 14.42 122.2 ± 10.92 11.49 108.64 ± 10.87 123.39 ± 13.5 13.57 121.64 ± 12.35 11.97

Total protein (mg/dl) 6.76 ± 0.59 6.92 ± 0.68 2.37 6.81 ± 0.69 0.74 7.03 ± 0.69 7.12 ± 0.69 1.28 7.08 ± 0.72 0.71 6.40 ± 0.62 6.97 ± 0.55 8.91 6.50 ± 0.62 1.56

Cholesterol (mg/dl) 66.4 ± 5.32 58.5 ± 5.70 −11.90 62.22 ± 6.21 −6.29 65.33 ± 6.22 57.0 ± 5.62 −12.75 61.83 ± 6.04 −5.36 67.0 ± 6.29 61.25 ± 6.15 −8.58 62.2 ± 5.54 −7.16

Triglycerides (mg/dl) 57.0 ± 5.68 52.2 ± 5.81 −8.42 54.80 ± 6.13 −3.85 59.6 ± 5.2 56.7 ± 6.11 −4.86 57.4 ± 6.58 −1.68 58.5 ± 5.91 58.1 ± 6.21 −0.68 58.37 ± 6.72 −0.22

HDLc (mg/dl)

LDLc (mg/dl)

42.73 ± 4.4 37.63 ± 3.81 −11.94 41.0 ± 4.44 −4.05 41.11 ± 4.6 38.02 ± 4.53 −7.52 40.0 ± 5.2 −2.70 42.7 ± 4.8 39.85 ± 4.46 −6.67 40.97 ± 5.21 −4.05

12.21 ± 1.23 10.0 ± 0.98 −18.09∗ 11.0 ± 1.11 −9.91 12.3 ± 1.19 9.25 ± 0.97 −24.79∗∗ 11.81 ± 1.22 −3.98 12.53 ± 1.22 9.0 ± 1.05 −28.17∗∗ 12.25 ± 1.13 −2.23

Lead (␮g/dl) 0.32 ± 0.05 0.48 ± 0.05 50.0∗∗ 0.33 ± 0.04 3.13 0.34 ± 0.06 0.52 ± 0.07 52.94∗∗ 0.37 ± 0.05 8.82 0.36 ± 0.06 0.56 ± 0.08 55.55∗∗ 0.38 ± 0.05 5.55

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VC + S + L: rats treated with lead and supplemented with Vitamin C and silymarin; N: number of rats in each group. a Percentage difference of controls. ∗ Significant at P < 0.05. ∗∗ Highly significant at P < 0.01.

Total lipids (mg/dl) 148.5 ± 11.1 133.8 ± 13.55 −9.89 136.42 ± 14.7 −8.13 149.53 ± 12.17 135.9 ± 13.8 −9.12 141.53 ± 15.66 −5.35 150.5 ± 13.2 137.82 ± 14.8 −8.43 144.3 ± 16.2 −4.12

M.G. Shalan et al. / Toxicology 206 (2005) 1–15

ALT (U/L) AST (U/L) GGT (U/L) ALP (U/L)

Difference (%) 12.33 8.00 5.13 1.19

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Fig. 1. Optical density of basal and apoptotic fragments of DNA at 180, 360 and 540 bp in liver of rat treated with lead acetate, Vitamin C and silymarin for 2, 4 and 6 weeks.

rats treated with lead acetate than controls, while Vitamin C and silymarin elevated the reduction of the intensity of DNA towards control. Lead acetate increases the intensity of apoptotic bands (Fig. 2) at 180, 360 and 540 pb with values of 22, 41, 53; 61, 84, 95; 82, 106, 133 after 2, 4 and 6 weeks of treatment than controls (16, 46, 63) respectively. On the other hand Vitamin C and silymarin supplementation increase the optical density towards the control with values of 19, 31, 41; 49, 80, 89; 66, 101, 132 at 180, 360 and 540 bp after 2, 4 and 6 weeks of treatment. The intensity of hepatic total RNA was decreased in rats treated with lead after 6 weeks of treatment (Figs. 3 and 4). Vitamin C and silymarin supplementation elevated the intensity of total RNA to approach to the controls. Fig. 2. Apoptotic DNA fragmentation in liver of rats treated with lead acetate and the protective role of Vitamin C and silymarin supplementation. Lanes 1 and 2: controls; lane 3: Vitamin C + silymarin + Pb for 2 weeks; lane 4: lead treatment for 2 weeks; lane 5: Vitamin C + silymarin + Pb for 4 weeks; lanes 6 and 7: lead treatment for 4 weeks; lane 8: Vitamin C + silymarin + Pb for 6 weeks; lanes 9 and 10: lead treatment for 6 weeks; M: 1 kp ladder.

3.3. Histopathological findings Histopathological findings showed that after 2 weeks of lead ingestion, mild focal hepatocyte proliferation manifested by thickening of liver cell plates, occasional binucleated cells and very few mitotic fig-

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Fig. 3. Optical density of electrophoretic RNA in liver of rats treated with lead acetate, Vitamin C and silymarin for 6 weeks.

ures occurred without any localized distribution among liver zones 1–3 (Fig. 5). Congestion of terminal hepatic venules (central veins) and portal vein branches and sinusoidal dilation (CVC) are evident (Fig. 6). In rats

Fig. 4. Gel electrophoresis showing the effect of lead acetate on electrophoretic pattern of RNA (EPRNA) of rats liver tissues after 6 weeks of treatment and the protective role of Vitamin C and silymarin supplementation. Lane 1: control; lanes 2 and 3: lead treated group; lanes 4 and 5: Vitamin C + silymarin + Pb group; M: 1 kp ladder. Liver specimens from rats treated with lead acetate for 2 weeks [group 1] (Figs. 5 and 6).

treated with Vitamin C and silymarin, hepatocyte proliferation was not evident, however, the liver showed mild venous congestion (Table 3). After 4 weeks of lead administration, mild chronic mononuclear inflammatory exudate comprising mainly lymphocytes infiltrated the portal areas. The infiltrate was localized within the portal region with no extension to adjacent hepatic parenchyma (no interface hepatitis). Tiny foci of spotty lobular necrosis and apoptotic bodies are found (Fig. 7). No evidence of hepatocyte proliferation, however, the congestion manifestations are prominent. Prolonged administration of lead for 6 weeks showed increased intensity of the portal inflammatory infiltrate (Fig. 8), breaching of the limiting plates by lymphocyte type of interface hepatitis (Fig. 9), frequent apoptotic bodies and foci of confluent necrosis (Fig. 10). Mild portal fibrosis with shot slender fibrous septa was found in all rats. Signs of hepatic congestion were exaggerated. Sinusoidal and portal macrophages laden with yellowish-brown pigment are seen (Fig. 11). Moderate macrovesicular steatosis of zone 3 was seen in one rat (Fig. 12). Remarkable reduction of the total histological activity index (portal and lobular) occurred when silymarin and Vitamin C were administered with lead.

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Fig. 5. Note proliferation of hepatocytes and thickening of liver cell plates without localized distribution among hepatic acinar zones 1–3; OM × 40.

4. Discussion Lead is dispersed throughout the environment, in ambient air, in many foods, in drinking water and in dust (Michael, 1997). The major environmental sources of metallic lead and its salts are paint, auto exhaust,

and contaminated food and water (Shy, 1990; Royce et al., 1990; Bornschein et al., 1985). There is evidence that some nutrients, especially Vitamin C, exhibit some protective effects against intoxication with lead (El-Zayat et al., 1996; Houston and Johnson, 2000).

Fig. 6. Sinusoidal dilatation is prominent; OM 200×.

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Table 3 Histopathological findings; HAI, and stage of fibrosis in different animal groups HAI

Fibrosis (0–6)

Apoptosis

Macrophages

Steatosis

0 0 0.25

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 1.4 0.4

0 0.4 0

0 + 0

0 0 0

0 0 0

0 1.75 0.25

0 5.25 1.25

0 1.75 0.25

0 ++ +

0 +++ +

0 + 0

P (0–4)

C (0–6)

S (0–4)

PP (0–4)

Total (0–18)

2 weeks Control Lead Lead + S + VC

0 0 0.25

0 0 0

0 0 0

0 0 0

4 weeks Control Lead Lead + S + VC

0 1.00 0.4

0 0 0

0 0.4 0

6 weeks Control Lead Lead + S + VC

0 2.75 1.00

0 0 0

0 0.75 0

HAI: histological activity index; P: portal inflammation (0–4); C: confluent necrosis (0–6); S: spotty necrosis (0–4); PP: periportal inflammation (0–4); (+) mild; (++) moderate; (+++) severe. Data represent mean values of each parameter.

This study clearly showed that lead acetate ingestion with a concentration of 500 mg/kg diet induced a significant elevation of serum ALT, AST, GGT and ALP levels as early as the end of the second week of treatment. Hassanin (1994) has reported that serum ALT

was elevated significantly more than AST on lead exposure. Elevated serum GGT has been observed in chronic hepatobiliary diseases indicating toxic liver damage (Bauer, 1982) and correlating with the development of fibrosis (Latner, 1975). Similarly, Tandon et al. (1997),

Fig. 7. Hepatic tissue of a rat treated with lead for 4 weeks [group 2]: a focus of spotty lobular necrosis infiltrated by mononuclear cells (yellow arrow). Two apoptotic bodies are seen (green arrows); OM 200×. Liver specimens of rats treated with lead acetate for 6 weeks [group 3]: Figs. 8–12.

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Fig. 8. A portal area infiltrated by mononuclear inflammatory cellular exudate comprising mainly lymphocytes. The limiting plate is branched by lymphocytic type of piecemeal necrosis (arrow). Proliferation of bile duct cells is also seen. The portal vein branch is congested and dilated; OM 200×.

Fig. 9. The limiting plate is disrupted by lymphocytic infiltration (interface hepatitis); OM 600×.

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Fig. 10. A focal area of focal confluent necrosis of hepatocytes (arrow). The sinusoids are dilated and congested. The portal inflammatory infiltrate is mild; OM 200×.

Fig. 11. Portal macrophages-laden with yellowish brown pigment; OM 200×.

El-Sayed et al. (1995) and Khan et al. (1993) reported disturbances in the liver functions after chronic lead exposure. We noticed no significant changes in serum total lipid, cholesterol, triglycerides and HDLc levels, however, serum LDLc decreased significantly in

all the studied groups. In accordance with our findings, Skoczynska et al. (1993) observed a decrease in plasma cholesterol level and lipoproteins in rats poisoned with small doses of lead. These results were also in agreement with the results of Antonowicz and Andrzejak (1996) that recorded no disturbances

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Fig. 12. Moderate macrovesicular steatosis especially at acinar zone 3; OM 100×.

in lipid metabolism in males exposed to lead pollution. It was shown that, lead nitrate induced a synchronized wave of hepatocyte proliferation in rat liver without accompanying histopathological necrosis in a dose-dependent manner. DNA synthesis was detected in a few scattered hepatocytes and in non-descript periductular cells. Proliferation on non-parenchymal cells reflect a direct mitogenic effect of lead nitrate and that hepatocyte proliferation follows the nonparenchymal cell reaction to lead nitrate (Rijhsinghani et al., 1993; Kubo et al., 1996). Recent studies have shown that lead-induced DNA damage (Fracasso et al., 2002; Valverde et al., 2002; Danadevi et al., 2003; Hengstler et al., 2003; Xu et al., 2003). In the present study, lead reduced hepatic total RNA content indicating a lower rate of hepatic protein synthesis. This observation was in line with the results of Hassanin (1994) and El-Zayat et al. (1996) who found a decrease in hepatic total protein content in response to lead intoxication. They attributed that to a decreased utilization of free amino acids for protein synthesis. Pagliara et al. (2003) showed that lead-induced liver hyperplasia followed by apoptosis mediated by oxidative stress in kupffer cells. Also, it induced apoptosis in the germ cells within the semineferous tubules

(Adhikari et al., 2001) and in rod photoreceptors (He et al., 2003). Iavicoli et al. (2001) demonstrated that the induction of apoptosis contributes to the Pb-induced inhibition of cell proliferation in rat fibroblasts. In contrast, Shen et al. (2001), Cheng et al. (2002) and De la Fuente et al. (2002) showed that lead as high as 500 ␮M did not induce apoptosis. Vitamin C was reported to reduce the possibility of lead interacting with critical biomolecules and thus preventing its toxicity (Hsu and Guo, 2002). Dawson et al. (1999) showed that daily supplementation with 1000 mg of ascorbic acid results in a significant decrease of blood lead levels, possibly by reducing the intestinal absorption of lead. It is of worth mentioning here that blood lead levels, in our study were lowered to that of normal controls after supplementation of intoxicated rats with Vitamin C and silymarin. Cheng et al. (1998) observed inverse associations of blood lead levels with total dietary intake of Vitamin C and iron. Some animal studies suggested that orally administered ascorbic acid may chelate lead and decrease the risk of its toxic effects (Simon and Hudes, 1999). Vij et al. (1998), Matte (1999), Houston and Johnson (2000) and Kleszczewska (2001) indicate a significant protective action of Vitamin C against toxic effects of lead on heme synthesis and drug metabolism. Marques et al.

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(2001) showed that co-treatment of lead with Vitamin C (3 mmol/l) prevented the increase of mean arterial blood pressure and restored the normal expression of endothelial nitric oxide synthase and soluble guanylate cyclase proteins in the vascular wall. The preventive activity of Vitamin C may related to its antioxidant efficacy that inhibits lipid peroxidation enhanced by lead (Upasani et al., 2001). On the other hand, Simon and Hudes (1999) suggested that elevated serum levels of Vitamin C may not associated with decreased elevated blood lead levels. However, Anderson and Yu (1994) showed that there were small protective effects of Vitamin C at low doses and exacerbating effects at high doses. Consistent with our results, Blankenship et al. (1997) showed that Vitamin C protected cells from undergoing apoptosis. A finding may be attributed to the combined action of Vitamin C and silymarin. This may associate with the antibiotic activity of silymarin through effect on transforming cell B and matrix gene expression of hepatic stellate cells (Fuchs et al., 1995). The results of the present study indicated that ascorbic acid and silymarin supplementation ameliorated the hepatic necroinflammatory lesions induced by lead ingestion. Silymarin has ant-inflammatory activities mediated by alteration of kupffer cell function (Dehmlo et al., 1996). It was reported that silymarin improved liver function tests related to hepatocellular necrosis and/or increases membrane permeability (Buzzelli et al., 1993). Ramadan et al. (2002) reported that the protective effect of silymarin was attributed to its antioxidant and free radicals scavenging properties. Germano et al. (2001) showed that silymarin clearly recovered histopathological changes produced by CCl4 , such as necrosis, fatty change, ballooning degeneration and inflammatory infiltration of lymphocytes around the central veins. Silymarin was found to reduce hepatic collagen accumulation by 35% in rats with secondary biliary cirrhosis (Jia et al., 2001). Results of Horvath et al. (2001) suggested that silibinin modulates the cellular immunoresponse and restores impaired liver function through its antioxidant capacity. It was found that feeding of animals on silymarin–phospholipid complex normalized lipid metabolism and inhibited atherosclerosis. Saravanan et al. (2002) showed that Vitamin C or/and silymarin were hepatoprotective and have antioxidant effect against ethanol intoxication. The hepatoprotective

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effect of silibinin may be attributed to its ability to scavenge oxygen free radicals and inhibition of liver microsome lipid peroxidation (Mira et al., 1994). In conclusion, it is assumed that combined supplementation with Vitamin C and silymarin highly protected rats from lead toxicity.

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