Efficacy of orally administered montmorillonite for ...

Applied Clay Science 103 (2015) 62–66

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Research Paper

Efficacy of orally administered montmorillonite for acute iron poisoning detoxification in rat Parisa Mirhoseini Moosavi a, Ali Reza Astaraei a, Alireza Karimi a, Mohammad Moshiri b, Leila Etemad c, Majid Zeinali d, Gholamreza Karimi b,⁎ a

Department of Soil Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Medical Toxicology Research Center, School of Pharmacy, Mashhad University of Medical Science, Mashhad, Iran Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran d Pharmacy School and Social Security Organization, Mashhad, Iran b c

a r t i c l e

i n f o

Article history: Received 16 April 2014 Received in revised form 3 November 2014 Accepted 5 November 2014 Available online xxxx Keywords: Geophagy Montmorillonite Iron toxicity Iron adsorption

a b s t r a c t Iron overload in the amount of 10–20 mg/kg of body weight can induce toxicity. Iron causes its toxic effects in a form of oxidative stress and inhibition of key metabolic enzymes. It seems that montmorillonite clay can reduce absorption of elemental iron by iron fixation in the gastrointestinal tract. The potential effect of montmorillonite in decontaminating the gastrointestinal tract was investigated based on serum iron concentration. In the present experiment, ferrous sulfate (100 mg/kg) was administered to Wistar male rats followed by oral gavage of montmorillonite suspensions at three different doses (0.5, 1.0 and 1.5 g/kg). Blood for determination of serum iron concentrations was drawn at 1 h after montmorillonite administration. The results showed that montmorillonite clay with doses of 0.5 and 1.0 g/kg were the best for significantly decreasing the elevated serum iron concentration compared to 1.5 g/kg (p b 0.05), probably through the proper adsorption of elemental iron. The activity of control and 1.5 g/kg treated groups diminished compared with other groups. Rats in the control group also defecated loser fecal matter than others. © 2014 Published by Elsevier B.V.

1. Introduction Geophagy, the habit of eating clay or earth, has been practiced by humans for a long period of time (Abrahams et al., 2006; Gichumbi et al., 2011; Hooda et al., 2004; Mahaney et al., 2000; Reilly and Henry, 2000). Examination of the diets of certain aboriginal tribes of South America, Central Africa, and Australia, showed that people use clay to keep away from getting stomachache, dysentery, and food infections (Droy-Lefaix and Tateo, 2006). Sailors traditionally carried out similar practice on board ships (Droy-Lefaix and Tateo, 2006). Geophagia has also been considered as a source of mineral nutrient supplementation such as calcium, magnesium, zinc, manganese and iron (Hooda et al., 2004; Tateo and Summa, 2007; Tateo et al., 2001), but many researchers have introduced soil consumption as a risk factor of anemia by decreasing metabolic iron absorption (Dreyer et al., 2004; Minnich et al., 1968; Tateo and Summa, 2007; Young et al., 2010, 2011a, 2011b). The human body can not directly increase iron excretion, so regular iron monitoring is done through the digestive tract. Acute iron toxicity is induced by ingestion of 10–20 mg/kg of elemental iron per kg of body mass (Tokar et al., 2013). Serious symptoms include alterations in ⁎ Corresponding author. Tel.: +98 51 38823255; fax: +98 51 38823252. E-mail address: [email protected] (G. Karimi).

http://dx.doi.org/10.1016/j.clay.2014.11.010 0169-1317/© 2014 Published by Elsevier B.V.

level of consciousness, persistent vomiting, hematemesis, diarrhea, hemodynamic instability, metabolic acidosis, lethargy, tachycardia, hypovolemia, shock and coagulopathy occurring in acute iron toxicity (Hosking, 1971; Manoguerra et al., 2005; Perrone, 2011). Oxidative stress and inhibition of key metabolic enzymes are two important mechanisms in iron poisoning (Papanikolaou and Pantopoulos, 2005; Tokar et al., 2013). Approximately 5000 cases of iron supplement poisoning occur annually in the United States, and most of them occur in children by consuming iron tablets, capsules and syrup or drop ingestion. According to American Association of Poison Control Centres report, iron is one of the leading causes of poisoning deaths in children b6 years old (Brown and Gray, 1955; Eshel et al., 2000; Litovitz et al., 1986; Manoguerra et al., 2005; Whittaker et al., 2002). The mainstay of acute iron toxicity management includes early gastrointestinal decontamination by gastric lavage or whole gut irrigation; chelating therapy with deferoxamine (or deferipirone) and organ failure treatment. Activated charcoal is a first-line treatment for poisonings and standard method of gastrointestinal decontamination but it has no good effect on iron absorption. Whole bowel irrigation also has not altered the clinical outcome of iron-poisoned patients (Eshel et al., 2000; Tennebein et al., 1991). Montmorillonite is a clay mineral with substantial isomorphic substitution of structural cations, which lead to negative charges on the basal layers. Exchangeable cations in the 2:1 layers balance the

P. Mirhoseini Moosavi et al. / Applied Clay Science 103 (2015) 62–66

negative charges generated by isomorphic substitution. The kinetics of cation exchange in montmorillonite is fast and the cations are easily exchanged with solute ions by varying the cationic composition of the solution (Bhattacharyya and Gupta, 2008). The technological properties of clays are directly related to their colloidal size and crystalline structure in layers, meaning a high specific surface area, optimum rheological characteristics and/or excellent sorptive ability and a high affinity to various heavy metal ions (Bhattacharyya and Gupta, 2008; Chantawong, 2004; Kraepiel et al., 1999; López-Galindo et al., 2007; Shirvani et al., 2006). Based on these characteristics, montmorillonite has been used in biomedicine and clinical therapies (Han et al., 2012) as well as to prevent the accumulation of heavy metals in organs (Han et al., 2012; Kim and Du, 2009; Wei et al., 2010). In the last decade few studies have been conducted in relation to iron adsorption by clay minerals in the gastrointestinal tract and their potential on preventing iron entry to blood (Hooda et al., 2004; Minnich et al., 1968); therefore the hypothesis of clay minerals as the most active part of soil was evaluated for the treatment of acute iron poisoning. Finally, because there is no known study regarding the efficacy of montmorillonite on iron absorption in acute toxicity, we made considerable effort to investigate the quantity of Fe adsorbed by montmorillonite in iron intoxicated animals. 2. Experimental 2.1. Montmorillonite purification and preparation Montmorillonite samples were obtained from raw bentonite. Raw bentonite was prepared by Ghaen Zarin Khak Company (with trade name of Zarin binder). The bentonite mine is located in the city of Ghaen in the South Khorasan province and it includes more than 80% montmorillonite and also has some impurities such as quartz, feldspars, halite, gypsum and mica. It is very important that quartz and feldspars, which have large amounts of silica, are removed from clay mineral. The mean particle size of the raw bentonite was 325 mesh (44 μm diameter). To remove unwanted microorganisms and organic matter from samples, thermal processing at about 350 °C was performed. A combination of purification methods were carried out including wet sieving sedimentation, centrifugation and ultrasonification. Purification carried out according to the method described by Ha Thuc et al. (2010) with some modifications. In order to separate the pure montmorillonite particles (particle size-fractions required: b 2 μm) from associated minerals (quartz, feldspar, etc.), 50 g of bentonite was suspended in 1000 mL of distilled water by laying the cylinder for 24 h only for separation of clay particles, then clay suspension was stirred again and the b 2 μm fraction of a clay with density 2.65 g/cc was collected after 4.5 h from a distance of 6.1 cm (Burt, 2004). The sand fraction (N20 μm) was separated from the silt fraction using a 20 μm sieve. The supernatant liquids containing clay-sized particles of b2 mm particles were siphoned into separate 100 mL centrifuge bottles. Chemical treatments were applied to remove impurities such as salts, gypsum and calcium carbonate from samples (Ha Thuc et al. (2010). Sodium acetate–acetic acid (pH 5) buffer solution was used to remove carbonates from samples (Duman and Tunc, 2009; Ha Thuc et al., 2010). At the final stage, purified samples were cation exchanged with sodium chloride to produce homogeneous interlayer cations. The purified and cation exchanged samples were then dried at 110 ° C for 24 h and ground to powder. The efficiency of purification was determined based on X-ray, particle size, cation exchange capacity (CEC) and ratio peak of the quartz/montmorillonite analysis pre and post experiments. For XRD analysis montmorillonite samples were characterized by X-ray diffraction (XRD, λCuKα = 1.54) using a Bragg–Brentano (θ, 2θ) and XRD pattern collected from 2θ = 4° to 60°. X-ray diffraction (XRD) measurement was performed using a Philips PW1800 model diffractometer.

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The XRD pattern of purified montmorillonite was given in Fig. 1. The morphology of the samples was observed by scanning electron microscopy (SEM). CEC determination was done with ammonium acetate (NH4OAc) method. The particle size distribution of purified montmorillonite clay was determined by laser granulometry to determine the efficiency in separating the small particles from the whole materials by CORDOUAN model of particle size analyzer in Central Laboratory of Ferdowsi university of Mashhad. 2.2. Animal study All animal experiments were approved by the Animal Care Committee of the Mashhad University of Medical Sciences. Twenty male Wistar rats, 250–270 g weight, were divided in four groups. All animals received 100 mg/kg body weight of ferrous sulfate by applying ferrous sulfate drops (FSD) (Mad Pharmaceutical Company, Iran) by oral gavage. Montmorillonite suspensions were prepared by mixing various amounts of clay in distilled water, gavaged to the rats at three doses (0.5, 1.0 and 1.5 g/kg). Animals were deprived of food 12 h before the test. Five minutes after iron administration, rats were orally gavaged with clay suspensions. The control group received only the iron solution in distilled water. The rats were lightly anesthetized with ether during blood sampling. Blood samples were collected from orbital sinus before and 1 h after iron gavage. Blood serum samples were separated by centrifuge. Serum iron level (SIL) was measured by Pars Azmoon Kit according to the instructions of the manufacturer. The absorbance was measured at 578 nm using enzyme-linked immunosorbent assay (ELISA) reader (Statfax 2100, USA). The stool consistency and activity scores of the animals were all monitored. Stool consistency was scored as follows: 1: constipation, 2: stool with normal consistency, 3: loose stool and 4: defecation incontinence. The activity score of animals was evaluated according to our previous work. Grade 1: no voluntary movements after painful stimulation, Grade 2: stretch movements after painful stimulation, Grade 3: stretch movements after stimulation and Grade 4: spontaneous normal mobility (Moshiri et al., 2013). All animals were evaluated by an individual unaware of the groups. 2.3. Statistical analysis Results are expressed as mean ± standard deviations and were analyzed with ANOVA and Mann–Whitney test by using Spss11.5 software. Statistical significance was set at p b 0.05 for all tests. 3. Results and discussion There was no significant difference among the mean SIL of different groups of rats before FSD gavages (p b 0.05). The average serum iron concentrations of the control group were significantly higher than SIL among rats treated with 0.5 and 1.0 g/kg of montmorillonite 1 h after FSD administration (Fig. 2). However, there was no significant difference between SIL of rats that received 1.5 g/kg montmorillonite and control groups (p N 0.05). The animal activity in control and 1.5 g/kg of montmorillonite groups diminished in comparison to others (Table 1). The animals of the control group also defecated looser and darker fecal matter than others. All groups treated by montmorillonite had normal light stools without any constipation (Table 1). Constipation is the most common adverse effect of activated charcoal (Osterhoudt et al., 2004) but in our experiments we did not observe any constipation, therefore it seems that montmorillonite in the concentrations given can be more effective against constipation, however, it could be hazardous to ingest far more clay than is necessary—eventual constipation will occur at high doses. If montmorillonite leads to constipation we can consider a

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Fig. 1. X-ray diagram of purified montmorillonite (mineral identified in sample included montmorillonite (M) and cristobalite (C)).

laxative for treatment as activated charcoal treatment. In order to prove these issues further experiments are warranted. The highest dose of montmorillonite (1.5 g/kg) was not as effective as the other doses (p N 0.05). This may be due to montmorillonite particle aggregation, overlapping and overcrowding which led to decreasing the available surface area of clay and iron chelating capacity. More likely the lower doses match the surface area of the gastrointestinal tract, coating the lining (Tateo and Summa, 2007), after the lining is coated, additional clay would not increase absorption of Fe from the blood. Also the activity of the rats was reduced at 1.5 g/kg dose. The reduced activity of the rats may be from too much clay in their gut.

According to our results, it is proposed that the soluble iron in the gastrointestinal tract with montmorillonite application (without its contents) decreased. However, this effect is not dose-dependent, therefore, montmorillonite proved to be effective in reducing SIL with doses of 0.5 and 1.0 g/kg. Two mechanisms may be contributed in this finding. 1) Iron (Fe+2) binds to the montmorillonite particles saturated with sodium which is preferred Fe2+ due to the highest exchange with sodium ions and this complex reduces the bioavailability of iron (Bhattacharyya and Gupta, 2008; Kraepiel et al., 1999; Schultz and Grundl, 2004; Srinivasan, 2011; Zhao et al., 2010). Iron adsorption to the montmorillonite clay mineral surfaces is suggested as the most important mechanism of the adsorption process in low pH (Bradbury and Baeryens, 2002; Schultz and Grundl, 2004) where Fe is soluble. In the smectite group minerals, particularly montmorillonite, different mechanisms of element uptake such as surface complexation and cation exchange have been observed under different conditions and are responsible for creating different reactions on the clay (Bradbury and Baeryens, 2002). In our experiments the results of purification methods showed that this method was efficient for preparation of high purity and high quality montmorillonite with ≤ 2.0 μm particle sizes and cation exchange capacity of 130 me/100 g. The remaining mineral in samples was primarily cristobalite (Fig. 1). 2) Adhesion of montmorillonite particles on gastrointestinal mucosal surface creates a mechanical protection inhibiting the iron absorption from the gastrointestinal tract Table 1 Activity and diarrhea score of iron intoxicated rats treated by montmorillonite (0.5, 1 and 1.5 g/kg) vs. the untreated control, 1 h after ferrous sulfate gavage.

Fig. 2. The comparison of serum iron level (SIL) of iron intoxicated rats, which were treated with montmorillonite (0.5, 1.0 and 1.5 g/kg) or the untreated control group, 1 h after ferrous sulfate gavage. The results are reported as mean ± SD; * p b 0.05.

Parameters

Control

0.5 g/kg Montmorillonite



Activity Stool consistency

2.4 ± 0.24 2.8 ± 0.2

4.0 ± 0* 2.0 ± 0.01*

3.8 ± 0.2 * 2.0 ± 0.01*

3.0 ± 0 2.0 ± 0.01*

The results are reported as mean ± SD; * p b 0.05.


(Hooda et al., 2004; Sayers et al., 1974; Seim et al., 2013; Young et al., 2011a, 2011b). However, the efficacy of this new decontamination method by using montmorillonite revealed that decreasing in Fe absorption could occur by adsorption of Fe as the clay particles mix with ingesta or by clay binding to the mucin layer of the intestine (Seim et al., 2013). In theory such a lining could reduce the availability of element however quantitative evaluations are not available (Tateo and Summa, 2007). Moreover clay as a barrier can keep Fe from being adsorbed by the body, therefore clay particles adsorbing Fe from the gastrointestinal tract and decreasing the bioavailability of Fe result to decontamination of the gastrointestinal tract and finally lead to decreasing in the movement of Fe from the gastrointestinal tract into the body. The majority of dietary non-haem iron enters the gastrointestinal tract the ferric form. However Fe 3+ is thought to be essentially non bioavailable and therefore it must first be converted to ferrous iron prior to absorption (Sharp and Srai, 2007), therefore in low pH (stomach) the molecular form of the iron is the Fe+2 which can adsorb by the clay particles. Clay is an effective factor in reduction of SIL, because iron may be adsorbed by the clay which in extreme cases could result in iron deficiency or chronic anemia (Minnich et al., 1968; Young et al., 2011a, 2011b). A world-wide relationship between geophagy and iron deficiency has been documented in several articles (Cavdar et al., 1983; Dreyer et al., 2004; Geissler et al., 1988; Johnson and Stephens, 1982; Mokhobo, 1986; Nchito et al., 2004; Prasad, 1991; Shapiro and Linas, 1985; Sunit Singhi et al., 2003; Tateo and Summa, 2007; Thomson, 1997). However, Harvey et al. (2000) believed that there is a positive effect of ingested soil on reducing iron toxicity in animals, due to the presence of pure clay such as montmorillonite in soil. Our experiments are consistent with Hooda and coworkers results (2002, 2004) that showed certain soil minerals may scavenge Fe at low pH (b4) where Fe is soluble. Decreased Fe absorption by the montmorillonite from the simulated gastrointestinal tract fluids was about 41% to 75% (Hooda et al., 2004). A similar observation was reported by Seim et al. (2013) using an in vitro digestion/ Caco-2 cell model based on ferritin formation. Ferritin formation was used as an index of Fe bioavailability. Results showed that ferritin formation in cells exposed to geophagic earths and clay minerals was significantly lower in soil samples in comparison with the cells exposed to white beans samples. This reduction occurred due to iron adsorption by the geophagic earth and clay minerals indicating an inhibition of Fe bioavailability. This fact was also reported by Pebsworth et al. (2013). Many studies in humans have documented the effect of geophagy on Fe absorption using Turkish (Arcasoy et al., 1978; Cavdar and Arcasoy, 1972; Minnich et al., 1968), Texan (Talkington et al., 1970) and South African (Sayers et al., 1974) geophagic earth. Johns and Duquette (1991) discussed the effect of the iron chelating organic compounds. They suggested that complex formation between organic materials and Fe resulted in adhesion of Fe to clay surface and reduced the bioavailability of iron. In our experiment, the iron concentration range and suspension composition was varied from those applied in other studies and we considered the effect of montmorillonite in an animal model of acute iron toxicity. Moreover some evidence indicates that montmorillonite is a non-specific adsorbent of different types of heavy metals, such as lead, arsenic and cadmium (Han et al., 2012; Kim and Du, 2009; Wei et al., 2010). Wei et al. (2010) have investigated the effect of montmorillonite on dietary lead accumulation in tissues of Tilapia fish using 0.5 wt.% montmorillonite in diet supplementation over 60 days. They observed that montmorillonite decreased the bioavailability of lead from tissues. Similar results were reported by Blanusa et al. (1989) and Han et al. (2012) who carried out an experiment on the effect of montmorillonite on accumulation of dietary As (III) exposure in tissues of common carp using 1 wt.% montmorillonite over 30 days. Kim and Du (2009) also explained the positive effect of 0.5 wt.% montmorillonite reducing the absorption of dietary Cd and its accumulation in organs of Cyprinus carp over 60 days of exposure.

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Activated charcoal is a non-specific adsorbent which is not actually a suitable material to remove heavy metals (Bhattacharyya and Gupta, 2008; Eshel et al., 2000). Therefore we can consider montmorillonite as a good adsorbent for iron toxicity (Eshel et al., 2000). Deferoxamine, also known as a Fe chelator compound, could not prevent absorption of ingested ferrous sulfate (Eshel et al., 2000). Eshel et al. (2000) noted that administration of activated charcoal and deferoxamine after 10 and 20 min did not show the Fe removal from the gastrointestinal tract in to the body. However, deferoxamine has significantly decreased SIL by increasing the excretion of iron from the body. It is noteworthy that even the use of deferoxamine has not been helpful in the reduction of Fe (II) absorption in volunteers or in acute Fe poisoning. In this study rats gavaged by the montmorillonite suspension 5 min after FSD gavaged. It seems that an appropriate dose of montmorillonite can reduce the SIL in rats poisoned with ferrous sulfate. We expected montmorillonite to have a great inhibitory on Fe uptake by the body however montmorillonite administration may not be effective in diminishing the serum iron level concentration over a long period of time considering the quick iron absorption in the body (Wheby et al., 1964). 4. Conclusion Our results show that orally administered montmorillonite in appropriate concentrations can relieve (it does not actually prevent Fe-poisoning, it is a treatment for Fe-poisoning) Fe-poisoning in this animal model. More studies are needed to evaluate the potential release of heavy metals from montmorillonite under gastrointestinal conditions, and to elucidate the accumulation of potential toxins in tissues and its effects on organs. Future studies should address the best time and duration of treatment by montmorillonite; in addition to the appropriate concentrations of the clay for treating humans. Acknowledgments The authors are thankful to the vice chancellor of Ferdowsi University of Mashahd, Mashahd University of Medical Sciences and Iran National Science Foundation for financial support. References Abrahams, P.W., Follansbee, M.H., Hunt, A., Smith, B., Wagg, J., 2006. Iron nutrition and possible lead toxicity: an appraisal of geophagy undertaken by pregnant women of UK Asian communities. J. Appl. Geochem. 21, 98–108. Arcasoy, A., Çavdar, A.O., Babacan, E., 1978. Decreased iron and zinc absorption in Turkish children with iron deficiency and geophagia. Acta Haematol. 60, 76–84. Bhattacharyya, K.G., Gupta, S.S., 2008. Adsorption of a few heavy metals on natural and modified kaolinite and montmorillonite: a review. Adv. Colloid Interf. Sci. 140, 114–131. Blanusa, M., Piasek, M., Kostial, K., 1989. Interaction of lead with some essential elements in rat’s kidney in relation to age. Biol. Trace Elem. Res. 21, 189–193. Bradbury, M.H., Baeryens, B., 2002. Sorption of Eu on Na and Ca- montmorillonite: experimental investigation and modelling with cation exchange and surface complexation. Geochim. Cosmochim. Acta 66 (13), 2325–2334. Brown, R.J.K., Gray, J.D., 1955. The mechanism of acute ferrous sulphate poisoning. J. Can. Med. Assoc. 73 (3), 192–197. Burt, R., 2004. Soil Survey Laboratory Methods Manual. Soil Survey Investigation Report. Nat. Resour. Conserv. Serv. 42, 1–700 (Ver .4.0.). Cavdar, A.O., Arcasoy, A., 1972. Hematologic and biochemical studies of Turkish children with pica: a presumptive explanation for the syndrome of geophagia, iron deficiency anemia, hepatosplenomegaly and hypogonadism. Clin. Pediatr. 11, 215–223. Cavdar, A.O., Arcasoy, A., Cin, S., Babacan, E., Gozdasoglu, S., 1983. Geophagia in Turkey: iron and zinc deficiency, iron and zinc absorption studies and response to treatment with zinc in geophagia cases. Prog. Clin. Biol. Res. 129, 71–97. Chantawong, V., 2004. Adsorption of Heavy Metals by Montmorillonite. The Joint International Conference on Sustainable Energy and Environment (SEE). Hua Hin, Thailand. Dreyer, M.J., Chaushev, P.G., Gledhill, R.F., 2004. Biochemical investigations in geophagia. J. R. Soc. Med. 97, 48. Droy-Lefaix, M.T., Tateo, F., 2006. Clays and clay minerals as drugs. In: Bergaya, F., Theng, B.K.G., Lagaly, G. (Eds.), Handbook of Clay Science. Elsevier, Amsterdam, pp. 743–752.

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