Precautionary role of lead poisoning chelation therapy in maintaining ...

INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCE AND HEALTH CARE ISSUE 2, VOLUME 2 (APRIL 2012)

ISSN: 2249-5738

Precautionary role of lead poisoning chelation therapy in maintaining a balanced Telomerase reverse transcriptase gene expression 1

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Shadia A. Fathey.; Abd El-Baset M.S.; 1Fatma F. Abd El Hamid; 2,3Bassem M.R. 4Sabah M. Hassan and 2 Noha A. Latif 1 2

Biochemistry Dept. Faculty of Science, Ain Shams University, Cairo, Egypt

National Research Center, Genetic Engineering and Biotechnology Division. 12622- Dokki, Giza, Egypt 3 4

College of Applied Medical Science, Taif University, Taif, KSA

Genetics Dep. Faculty of Agriculture, Ain Shams University, Cairo, Egypt

Corresponding author Bassem M. Raafat Assistant Professor of Biophysics Genetic Engineering and Biotechnology Division, National Research Center, Cairo, EGYPT 3

College of Applied Medical Science, Taif University, Taif, KSA 002 0128 0887880 – 00966 56 888 3760 [email protected]

ABSTRACT Environmental pollution caused by heavy metals is responsible for numerous pathologies in wild species. Lead has been the cause of serious risks to man specially in developing countries. This study was aiming to monitor the effect of elevated lead ions concentration on the telomerase enzyme activity as well as finding out to what extend chelation therapy may overcome these effects. Animal study design was conducted to sixty male Sprague-Dawley rats. Animals were divided into four groups, each contains 15 rats:: GroupI: Negative control group which represents rats did not receive lead through drinking water, Group II: Positive control group which represents rats received lead acetate (1000 ppm) through drinking water for 21 day. GroupIII : rats received lead acetat in drinking water for 21 days and then receive extract of Angelica Archangelica roots for another 21 days with continuous exposure to lead acetate in water. GroupIV: rats received lead acetate in water for 21 and then receive doses of dimercaptosuccinic acid (DMSA) for another twenty one days with continuous exposure to lead acetate in water. Lead ions concentration in blood and liver was measured as well as telomerase enzyme activity in liver. Histopathological examination of liver of all groups was carried out. Rsults indicate that exposure of rats to lead actate decreased the telomerase enzyme gene expression driving the cells to increase the apoptotic rate. Treatment with Angelica Archangelica root extract enhanced the gene expression and gave the best results when the treatment started from the beginning of the poisoning process. Keywords: lead intoxication – DMSA – Angelica Archangelica – telomerase enzyme activity.

Introduction: Lead is an environmental neurotoxicant known to produce detrimental effects in the nervous system [24]. Chronic exposure to lead leads to its accumulation in vital organs with maximum concentration reported in kidneys [16]. Developing brain is particularly vulnerable to the toxic effect of lead resulting in number of behavioral abnormalities including learning impairment, decreased hearing and impaired cognitive functions in human and experimental animals [44]. In the majority of cases, children with lead poisoning are asymptomatic resulting in a delay in the appropriate diagnosis [17]. However, during this time effects on a cellular level are occurring resulting in subtle

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changes in the child. These include impairment of IQ and other cognitive effects, decreased heme synthesis, and interference in vitamin D metabolism [36]. Johnson [18] indicated that the lead treatment is coupled with enzyme inhibition, fidelity of DNA synthesis, mutation, chromosome aberrations, and cancer. Chelation therapy may be used to decrease the blood lead concentrations. Several chelating agents are effective in lead excretion, but the chelator of choice depends on the blood lead concentration, the patient‟s symptoms and the environmental lead burden [26]. 2, 3-dimercaptosuccinic acid (DMSA) and dithiol- containing metal chelator were the first orally administered chelator approved by the FDA for treatment of childhood Pb poisoning [27]. DMSA may be used to reduce blood and tissue lead levels [35]. Chelating agents bind metals at two or more sites. Ideally, the chelated metal would be excreted; however, the lead-chelate complex may persist in tissues where the binding occurred or be redistributed to other tissues [26]. The chelation potency of Angelica Archangelica roots extract was approved by administrating this extraction with different concentrations as an oral chelator in rats received lead acetate in water. Blood lead concentration was dramatically reduced after administration of this extract for twenty one day after complete stoppage of more lead poisoning process. Lead ions concentration also decreased in both liver and kidneys. No significant change was detected regarding bone lead concentration after treatment [7]. Telomeres are DNA–protein structures that are localized at the ends of eukaryotic chromosomes composed of hexanucleotide repeats, (TTAGGG) n [12]. In normal human somatic cells telomere shortening has been observed both in vitro with cell replication and in vivo with aging. Telomere length has been shown to predict replicative potential of human somatic cells, since, in the absence of compensatory mechanisms; telomeres shorten with each cell division until reaching a critical length where further replication cannot occur [11]. In most organisms the main mechanism of telomere length maintenance is completion of DNA telomere repeats by telomerase [13]. This enzyme elongates the chromosome 3-end, whereas the complementary strand is completed by DNA polymerases [47]. Telomerase is a ribonucleoprotein complex. Telomerase contains two components, an RNA component and a catalytic subunit referred to as the telomerase reverse transcriptase (TERT) [30]. Some of these components are necessary for telomerase attachment to the telomere at a certain cell cycle phase [32], while others are required for regulation of telomerase activity [15]. Some proteins are necessary for maturation of the telomerase complex and degradation of its components [4]. The amount of telomerase in the different types of cells undergoes fine regulation [31].This is important because telomere shortening in human cells and finally senescence will result in restriction of cell division potential [5]. The cell protective function of telomerase has been related to improved DNA damage repair [42] or to increased apoptosis resistance [28]. It had been shown that telomerase is excluded from the nucleus upon oxidative stress [37]. Stress-dependent nuclear exclusion has been demonstrated not only for ectopically overexpressed TERT, but also for wild-type TERT in endothelial cells, indicating that this shuttling is not an overexpression artifact [14]. Santos et al. [38] found an aggravation of mitochondrial DNA (mtDNA) damage in cells expressing TERT. Although telomere length reduction and following initiation of senescence is the usual fate of somatic cells, it is possible that the lack of certain telomerase components results in early phenotypic senescence with loss of function at the cellular and system levels [10]. The aim of this work was to evaluate the effects of lead poisoning on rats by investigating the blood and liver lead levels and histopathological liver changes to make a correlation between these parameters with telomerase enzyme activity as well as the evaluation of the potential reversibility of these effects after chelation therapy using Angelica Archangelica root extract and dimercaptosuccinic acid (DMSA).

Materials and Methods:

Animals and treatments: The experiments were performed on sixty male Sprague-Dawley rats weighing 200 g (±0.1 kg). All through the experiment duration, the rats were housed in separate cages, fed standard laboratory food and allowed free access to water in room lightening with a 12 hour light-dark cycle in animal house of National Research Center (NRC). Experimental animals design was priory approved from the National Research Center ethical committee.

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Animals were divided into four groups each contains 15 rats: Group I : Negative control group which represents rats did not receive lead acetate through drinking water, Group II : Positive control group which represents rats received lead acetate (Pb (CH3COO)2. 3H2O El-Nasr Pharmaceutical Chemicals CO.; 1000 ppm) through drinking water for 21 day. GroupIII: rats received 1000 ppm lead acetate in water for 21 day and then received Angelicaarchangelica roots water extraction, as natural chelator, three times per week for 21 day with continues exposure to lead. Group IV: 15 rats received 1000 ppm lead acetate in drinking water for 21 day and then a dose of dimercapto succinic acid (DMSA), as a chemical chelator, three times per week orally for another 21 day with continues exposure to lead. Drinking water was prepared as 1000 ppm lead acetate solution. Acidification of water is essential for dissolving lead acetate in water so 1 ml of conc. hydrochloric acid was added per liter of deionized water 1000 ppm lead acetate dose is chosen to give a desired level of toxicity in blood and other soft tissue pools. Angelica archangelica roots were crushed. Boiled distilled water was added to 15gm of this powder. After cooling, juice was filtered three times using Wattman filter no. 4. The obtained solution was used in a base of body weight as 5 ml for each kg body weighs [2]. 50 mg/kg body weight of DMSA was orally administered to rats [21]. Blood samples were collected, after 21 day for group I and II and after 42 day for group III and IV, for the biochemical and molecular investigations by a suitable syringe for this purpose and stored in tubes containing heparin at -70°C until the time of analysis. Determination of lead concentration in rat tissues: Determination of lead concentration in rats tissues was performed using dry ashing technique of tissue specimens were obtained at the time of euthanization including, blood and liver. Lead concentration was determined by atomic absorption with a graphite furnace utilizing modified methods of Yeager et al. [47]. Histopathological examination: At the end of the experimental period and after the collection of blood samples, the rats were sacrificed with intravenous injection with sodium pentobarbital and subjected to a complete necropsy. Representative samples of liver were fixed in 10% neutral buffered formalin for routine histopathology. Sections were cut in 5 mm and stained with haematoxylin and eosin (H&E) using standard protocol. The transmission electron microscope images of rats liver were performed in the National Cancer Institute using Jeol jem-100s electron microscope.

Total RNA extraction: RNA was extracted by simply P spin column total RNA extraction Bioer kit package, Cat. No.BSC52S1 (Hang Zhou Bioer Technology Co., Ltd, Chaina). The tissues ( 0.05) compared to GI c significant difference (P > 0.05) compared to GII

Table 1 shows that administration of lead acetate for 21 days increased both liver and blood lead ions compared to negative control group (GI)( P>0.05). Dramatic significant reduction was recorded in rats received chelator but Angelica extraction showed higher chelation potency regarding lead ions in both blood and liver (12.12 ± 1.47 and 23.47 ± 1.98 µg/dl, respectively). Attention should be drawn to blood and liver lead concentration in rats treated with DMSA; its reduction value was lower than that recorded in rats treated with herbal chelation (64.14 ± 5.14 vs 23.47 ± 1.98). Histopathological examination: Microscopic examination of hepatocytes and their nuclei revealed that, normal liver of negative control group in which rats did not receive neither lead acetate in the drinking water or any treatment are shown in figures 1A and 1B. Liver transverse sections after lead acetate administration for 21 days showing severe hydropic and vascular degeneration were seen in the hepatocytes mainly periphery of the centrilobular zone (Figure 1C). In these areas, hepatocytes were swollen, pyknosis, vascular and pyknotic. The central veins as well as portal veins engorged with blood partially the central vein (Figures, 1C&2D). A clear dilatation and congestion of the portal veins was noticed. In addition, perportal necrosis of the hepatocytes that surround the portal areas and the inflammatory infiltration was seen in Figure 1D. Microscopic examination of rats liver exposed to 21 days of lead in drinking water followed by another 21 days of DMSA treatment showed low level of pyknosis in the hepatocytes nuclei as shown in Figure 1E. Moderate vaculeation in the cytoplasm of hepatocytes could be counter. The portal area apparently showed no changes in rats treated with Angelica root extraction after twenty one days of lead exposure in the drinking water as in Figure 1F. It was very noticeable that rats received Angelica or DMSA showed lower effects as compared to those of lead acetate treated group.

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Figure (1): Microscopic examination of rat liver transverse sections. (A and B): Normal liver of negative control group in which rats have not receive neither lead acetate nor any treatment in the drinking water. (C and D): After lead acetate administration for 21 days, (E): After 21 days exposure to lead acetate in drinking water followed by another 21 day of lead acetate and DMSA treatment, (F): After 21 day of exposure to lead acetate in drinking water followed by another 21 day of lead acetate and Angelica root extraction.

Effect of lead administration on telomerase activity: To investigate the effect of the different treatments mentioned above on telomerase activity, real-time PCR measurement of rat TERT transcripts was performed to determine whether the transcript abundance for this critical telomerase subunit is altered by lead administration and other treatments or not. The mRNA expression for rat telomerase reverse transcriptase (rTERT) was detected where there are two different concentrations of mRNA were used. rTERT mRNA was not detectable using 50 ng/µl RNA concentration (recommended by the manual of RT-PCR quantification kit package Cat. No. BSB04M1). For this reason RNA another concentration was chosen (800 ng/µl). Although it is a very high RNA concentration, it gave very low gene expression levels for all groups with different cut off points. However the expression of rTERT in the negative control group was higher than the expression in lead acetate treated tissues. Meanwhile the expression levels after lead acetate in drinking water concomitant with both Angelica extract and DMSA chelators were lower than the negative control group and higher than the lead acetate treated group. Curve of the control group was the first curve to be appeared at cycle 35, whereas the curve of the lead acetate exposed group was the last curve to be appeared at cycle 38. This behavior indicates that the expression of rTERT is decreased by the exposure to lead compared to negative control and chelators have the potential to decrease the lead poisoning (Figure2).

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Figure (2): mRNA expression plot for the catalytic subunit telomerase reverse transcriptase (TERT) with two different concentrations of RNA (50 ng/µl and 800 ng/µl) in negative control group, positive control group (rat received lead acetate in drinking water for 21 days), as well as those treated by Angelica root extraction and DMSA with continuous exposure to lead for another 21 days.

Discussion: Human activities in industry and the increasing use of metals in industry has led to serious environmental pollution through effluents and emanations of large quantities of metals to drinking water. Lead is a persistent metal, however, and is still present in the environment in water, brass plumbing fixtures, soil, dust and imported products manufactured with lead [22]. Children are continuing to bear the burden of sensitivity to lead exposure. The diagnosis of lead toxicity has traditionally been based on significantly elevated blood lead levels. However, data now indicates that low-level exposures resulting in blood lead levels below 10 μg/dL result in cognitive dysfunction, neurobehavioral disorders, neurological damage, hypertension, and renal impairment. While chelation is the convention recommendation for acute lead toxicity with encephalopathic damage, treatment for low-level, chronic exposure is still under investigation. Issues surrounding the assessment of body lead burden and the consequences of low-level environmental exposure are critical in the treatment of chronic disease related to lead [33]. In the present study the obtained data showed significant increase in both blood and liver lead concentrations in lead acetate treated rats compared to negative control group. Liver from lead acetate treated rats also revealed several histopathological legions related to apoptosis. Apoptosis signs are very clear in liver cells of rats received lead acetate in drinking water with no treatment application. One of the most notable observations in the present study was the signiﬁcant depletion of blood and liver lead in rats received either Angelica or DMSA root extract as a chelator after lead acetate administration in drinking water for 21 day as compared to those received lead acetate. In addition, it was very noticeable that histopathological liver examination of rats received Angelica or DMSA with continues exposure of lead intoxication

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in water showed less changes compared to those of lead acetate treated group. These results are in agreement with the fact that, chelation therapy is the preferred medical treatment for reducing the toxic effects of metals. Chelating agents are capable of binding to toxic metal ions to form complex structures which are easily excreted from the body removing them from intracellular or extracellular spaces [9]. Although the concept of chelation is based on simple coordination chemistry, evolution of an ideal chelator and chelation therapy that completely removes specific toxic metal from desired site in the body involves an integrated drug design approach. Chelating agents are organic or inorganic compounds capable of binding metal ions to form complex ring-like structure called „chelates‟. Chelating agents can affect metal toxicity by mobilizing the toxic metal mainly into urine. A chelating agent forming a stable complex with a toxic metal may shield biological targets from the metal ion, thereby reducing the local toxicity [1]. Increasing the cell apoptosis rate is obviously shown in the histopathological examination demonstrated in figure (1). Apoptosis signs are very clear in liver cells of rats received lead acetate in drinking water with no treatment application. Again, best results were obtained when treatment was concomitant with the very beginning of the intoxication application. Telomerase is a reverse transcriptase well recognized for its function at telomeres, protein-DNA structures essential for the stability of linear chromosomes. The enzyme is composed minimally of two different subunits, a catalytic core (TERT) responsible for DNA catalysis and an RNA component (TERC) that is used as template for telomeric DNA synthesis. Whereas the RNA is present in both somatic and germ cells constitutively, expression of TERT is tightly regulated. Telomerase activity is high during embryogenesis and in the vast majority of tumors but is low or non-existent in most adult somatic cells [43]. Both TERT mRNA and protein have been reported in telomerase-negative cells albeit at low levels [20, 29] In accordance with the previous investigations, mRNA expression level of the catalytic subunit telomerase reverse transcriptase (rTERT) was very low in the different groups. Two different concentrations of RNA (50 ng/µl and 800 ng/µl) were used in all mentioned groups. The data showed that, there was no detectable rTERT mRNA when 50ng/µl (recommended concentration) was used. The level of rTERT gene expression was very low in all rats especially those received lead acetate in drinking water. Gene expression begun to appear when a very high RNA concentration (800 ng/µl) was used. However the expression of rTERT in the negative control group was higher than the expression in the lead acetate treated group. Meanwhile the expression levels after lead acetate in drinking water concomitant with either Angelica extract or DMSA chelators were lower than that in the negative control group and higher than that in the lead acetate treated group. These findings verify that, lead poisoning decrease the gene expression that in role may result in low gene repair after cell proliferation. This DNA repair weakness will drive the cells to go through programmed cell death. Previous studies have shown that Telomeres demonstrated high sensitivity to damage by oxidative stress due to their high content of guanines [19]. In addition, oxidative damage to nucleobases has been reported to accumulate over the life span of a cell or an organism, contributing significantly to senescence (ageing) due to the shortening of telomeres. Senescent cells were found to contain 30% more oxidative modified guanine in their DNA and four times as many free 8-oxod G-bases (8-oxo-deoxyguanosine) [45]. In contrast to the majority of genomic DNA, telomere DNA was reported to be deficient in the repair of single-strand breaks [34]. Telomerase function is regulated by genetic, epigenetic and environmental factors. Although most genes involved in telomere biology are highly conserved between species and have limited genetic diversity in humans [40], several polymorphisms in the TERT promoter region and genes coding for telomerase associated proteins have been shown to regulate telomerase gene expression and activity level [3,39]. Chronic life stress is linked to shorter telomeres and has been associated with reduced telomerase activity [8]. Numerous in vitro studies demonstrate that oxidative stress and inflammation reduce telomerase activity and shorten telomere length in cultured endothelial progenitor cells, human-derived fibroblasts and neoplastic cells [25, 41, 6, 23]. Another important finding comes from the correlation between chelation therapy of lead treated rats with Angelica root extract and DMSA on one side and the gene expression cut off on the other side. Enhancement of TERT expression was found in rats received chelating treatment. Attention should be drawn to the best results obtained when chelation therapy begun from the starting intoxication process. In conclusion, lead poisoning increased blood and liver lead concentration, cause liver damage and decreased the telomerase gene expression driving the cells to increase the apoptotic rate. The treatment with

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Angelica root extract and DMSA signiﬁcantly decreased lead levels in rat blood and liver samples, provided protection against lead-induced cellular liver damage and reversed almost all the damage to classic liver architecture and enhanced the gene expression. Angelica root extract and DMSA treatment gave the best results when treatment started from the beginning of the poisoning process.

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