Effect of arsenic trioxide along with tannic acid , disodium hydrogen phosphate , alum and effects of sand-charcoal-iron-filter bed filtrated water along with alum on body weight and some hematological parameters in rabbit

The effect of arsenic trioxide along with tannic acid, di-sodium hydrogen phosphate (DSHP), alum and effects of sand-charcoal-iron-filter (SCIF)-bed filtrated water along with alum on body weight and some hematological parameters were carried out on a total of 30 (01 month old) adult Newzealand white rabbits. The Rabbits were randomly divided in to 6 equal groups (A,B,C,D,E & F) at the ratio of three males and two females in each group, rats of group A was kept as control without giving any treatment, rabbits of group B received arsenic trioxide@100 ppm, group C received arsenic trioxide @ 100 ppm plus tannic acid @100 ppm, group D received arsenic trioxide @ 100 ppm plus di-sodium hydrogen phosphate @100 ppm, group E received arsenic trioxide @100 ppm plus alum @100 ppm orally daily for 60 days in all cases and group F received alum @100 ppm in SCIF-bed filtrated water orally daily for 60 days. The result showed that body weight gain of control group (A) per week per rabbit was found to increase but in treated group B (arsenic trioxide@100ppm orally daily) the mean body weight of group B were decreased gradually and significant (P<0.05) decrease in body weight was observed at 40 days of feeding and at day 60 it was highly significant (P<0.01). Rabbits of group C, D and E were apparently normal and mild body weight reductions were observed which was statistically insignificant. No body weight loss was observed in rabbit of group F. TEC, Hb and PCV were slightly reduced in group C, D, E and F but highly significant (P< 0.01) reduction of TEC, Hb and PCV were observed in group B. ESR values were slightly increased in group C, D, E and F but highly significant (P< 0.01) increased of ESR value was observed in group B. Leukocytosis was observed in rabbits of group B. In this study, it was concluded that treatment with arsenic oxide at low doses has harmful effects on experimental animals including hematological alterations.


Introduction
Arsenic (As) is -The Silent killer‖ for Bangladesh at present.In world water day 1998 arsenic marked as the king of poisons.Contamination of arsenic in Bangladesh is mainly geological.Now how this As comes up in drinking water is not fully understood but the experts opinion suggest that during the dry season (October-April) the water level of the country goes down due to non-recharge and excessive extraction of ground water every year but when the lost water is not replaced by rain water.The ground water contamination by As in Bangladesh was first identified in 1993 at Chapai-Nawabgonj district.The arsenic disaster of Bangladesh has been called the most terrible environmental catastrophe of the twentyfirst century.WHO described the condition as -the largest mass poisoning of a population in history‖ (WHO, 2001).About 85 million people are at risk of drinking arsenic contaminated water and foodstuffs (Hossain, 2006;Wahidur, 2006).In a recent report, Chakraborti et al. (2010) showed that hand tube wells of the tableland and hill tract regions of Bangladesh are primarily free from arsenic contamination, while the flood plain and deltaic region including the coastal region are highly contaminated with arsenic.The arsenic contamination of drinking water may also be due to industrial pollution.In Bangladesh, the arsenic concentration in drinking water is alarmingly higher than the standard set by WHO.Together with the poor socioeconomic and nutritional status of the population, the chronic exposure to arsenic in drinking water is causing widespread health hazards in both man and animals in Bangladesh.Drinking water normally contains inorganic arsenic as arsenate (As(V)) and arsenite (As(III)).Inorganic arsenic is more dangerous than many other toxic substances.It is four times as toxic as mercury.Anyone who drinks arsenic in water at 60 parts per million (ppm) will soon die.But, organic arsenic in food is less toxic than inorganic arsenic.Most of the ingested arsenic is excreted from the body through urine, stool, skin, hair, and nail.Nonetheless, if the ingestion of arsenic through drinking water is very high, then our body normally cannot escape its toxic effects.Arsenic is deposited in tissues and causes oxidative stress to cells resulting in multiple organ dysfunctions.Arsenic poisoning has previously been considered as one of the most common causes of toxicity in domestic animals and birds (Selby et al., 1977;Doyle & Spaulding, 1978;Jubb et al., 1985;Bazargani et al., 2007).Water and forage containing high concentrations of arsenic are the main ways through which arsenic enters the animal body, consequently gets into the human food chain, and may cause poisoning in humans and animals.Arsenic remains one of the most important carcinogens and diabetogenics in human (Eisler, 1988;Biswas et al., 2000;Mukherjee et al., 2004;Rana et al., 2010).Nowadays it is, after lead considered the most common toxic heavy metal affecting domestic animals (Selby et al., 1977;Doyle & Spaulding, 1978;Bazargani et al., 2007).Arsenic poisoning may occur as peracute, acute, subacute or chronic form.While sudden death without particular clinical signs may occur in the peracute form of poisoning, acute and subacute forms are associated with severe gastroenteritis.Chronic poisoning causes ill thrift, weakness and inability, milk reduction and abortion (Selby et al., 1977;Bazargani et al., 2007;Radostits et al., 2007).Presence of this metal in water has been reported from different countries such as USA, Canada, Mexico, Argentina, Chile, Poland, Japan, China, Taiwan, Nepal, Vietnam, Bangladesh, India and Iran (Wang et al., 2002;Mandal & Suzuki, 2002;Ng et al., 2003;Hosseinpourfeizi et al., 2007;Mosaferi et al., 2008;).Several cases of chronic arsenic poisoning in humans have been reported from Iran (Hosseinpourfeizi et al., 2007;Mosaferi et al., 2008), there is, however, only one animal report from an industrial cattle farm (Bazargani et al., 2007).In Bangladesh there is no available data in this context, so this research work has been carried out to study the effect of arsenic trioxide along with tannic acid, DSHP, alum and effects of SCIF-bed filtrated water along with alum on body weight and some hematological parameters in rabbit.

Experimental animals
One month old thirty apparently healthy adult Newzealand white rabbits (Oryctolagus cuniculus) weighing between 250-450 g were purchased from a local private farm of Muktagacha, Mymensingh, Bangladesh and brought to the Experimental Pharmacology and Toxicology laboratory at Bangladesh Agricultural University (BAU) for the present study.After two weeks of acclimatization animals were segregated on the basis of their age and body weight without significant differences.They were housed throughout the entire period of study in well ventilated animal house at a room temperature of 23 ±1 0 C and were supplied with standard ration formulated by ICDDRB, Dhaka and supplied fresh water ad libitum.

SCIF -bed filtered water
Sand-Charcoal-Iron-Filter (SCIF) bed was developed and used as arsenic purifying system.The artificially As contaminated water was passed sequentially four times through SCIF bed.The filtrated water was collected and examined by using Merck Arsen test kit and was used in the study.

Experimental design
The Rabbits were randomly divided in to 6 equal groups (A,B,C,D,E & F) at the ratio of three males and two females in each group, rats of group A was kept as control without giving any treatment, rabbits of group B received arsenic trioxide@100ppm, group C received arsenic trioxide@100ppm plus tannic acid@100ppm, group D received arsenic trioxide@100ppm plus di-sodium hydrogen phosphate@100ppm, group E received arsenic trioxide@100ppm plus alum@100ppm and group F received alum@100ppm in SCIF-bed filtrated water orally daily for 60 days in each cases.Prior to segregation, initial body weight of each rabbit was recorded and kept group wise in cages.After treatment all the rabbit were kept under close observation for a whole period of study and all the parameters (body weight gain or loss, hematological parameters eg.TEC, TLC, Hb%, PCV and ESR was recorded before and during treatment at specific time intervals.

Measurement of body weight
The body weight of each rabbit was measured just before starting of treatment and body weight gain or loss was recorded in each 10 days interval up to sacrificing of the animals.

Procedures for the collection of blood sample for measuring hematological parameters
Blood was collected just before treatment i.e. day 0 and during treatment on day 20, 40 and day 60 directly from marginal ear vein of rabbit.Immediately after collection blood was transferred to sterile tube containing anticoagulant (4% sodium citrate solution) at a ratio of 1:10 and used for determination of different hematological parameters.

Determination of total erythrocyte count (TEC)
The blood was sucked by the red pipette up to 0.5 mark of the pipette.Then the tip of the pipette was placed in to red cell diluting fluid and the pipette was filled with the fluid up to 101 mark.The contents of the pipette were mixed thoroughly by shaking with 8-knot motion for 3-5 minutes.The counting chamber was placed with cover glass under microscope using low power (10X) objective and a small drop of fluid was placed properly on the counting chamber.The cells were counted from the recognized 80 small squares under high power objectives (40X).After completion of counting total cells, the number of RBC recorded from the supplied samples were expressed as, No. of cells counted x 10,000 and the result was expressed in million/mm 3 .

Determination of total leukocyte count (TLC)
Well mixed blood was drawn up to 0.5 mark of white blood cell pipette.The diluting fluid (N/10 HCL) was filled up to the 11 mark of the pipette and the contents were thoroughly mixed for 2 minutes.The counting chamber was then placed properly and filled with one drop of fluid and examined under low power (10X) objective.The leukocytes in the four large squares (each 1 square mm) of the counting chamber were counted.The number of WBC was calculated as follows: No. of WBC= No. of cell counted x 50.The result was expressed in thousand /mm 3 .

Determination of hemoglobin content (gm%)
The N/10 HCL solution was taken in a graduated diluting tube up to 2 marks with the help of a dropper.Citrated well homogenized blood was then drawn in to Sahli pipette up to 20 µl mark.The blood of the pipette was immediately transferred in to the diluting tube containing HCl solution.This blood and acid were thoroughly mixed by a glass stirrer in to the diluting tube so that acid hematin mixture is formed.The tube containing acid hematin mixture was kept standing in the comparator for 5 minutes and distilled water was added drop by drop.The solution was mixed well with a stirrer untill the color of mixture resembled the standard color of the comparator.The result was read in daylight observing the height of the liquid in the tube considering the lower meniscus of the liquid column.The result was then expressed in gm%.

Erythrocyte sedimentation rate (ESR)
The fresh anticoagulated blood was taken in to the Wintrobe hematocrit tube by using special loading pipette exactly up to 0 mark.Then the filled tubes were placed vertically undisturbed the wooden rack for one hour and then ESR was recorded from the top of the pipette and expressed in mm in first hour.

Packed cell volume (PCV)
Blood filled Wintrobe hematocrit tube was centrifuged for 30 minutes@3000rpm.Then the hematocrit or PCV was recorded, the percent volume occupied by the hematocrit was calculated by the following formula as described by Coffin (1955).PCV% =Height of the red cell volume in cm/Height of total blood in cm x 100

Statistical analysis
Collected data were statistically analyzed by the computer using statistical package programme MSTAT-C developed by Russel (1996).A one way ANOVA was made by F variance test.

Effect on body weight
The mean body weight of rabbits of group F (+9.15%) significantly increased as like control group (group-A) (+6.79%) which was statistically significant (P<0.01).In group B significant decreased of body weight was observed on 60 days of arsenic trioxide feeding which was statistically highly significant (P<0.01).The body weight of group C, D and E was slightly increased on 60 days of arsenic trioxide feeding in combination with tannic acid, DHSP and alum respectively but were statistically not significant (Table -1).The mean body weight of group B were decreased gradually and significant (P<0.05)decrease in body weight was observed at 40 days of feeding and at day 60 it was highly significant (P<0.01).The similar result on body weight was observed by many other workers (Byron 1967;Suren, 1977;confer 1980).Rabbits of group C,D and E were apparently normal and mild body weight reduction were observed at the last part of the experiment which was statistically insignificant, this might be due to the interaction of arsenic oxide with other chemicals like tannic acid, DSHP and alum used in those groups respectively.No loss of body weight in rabbits of group F were observed during the whole experiment period might be due to supplied SCIF bed filtrated water with alum which indicates the successful use of SCIF bed to filtrate arsenic contaminated water as arsenic purifying system.The results of this study was in agreement with the findings of a research conducted by Khan et al.,2014 on mice, ducklings and broiler chicken in Pakistan.

Effect on hematological parameters 3.2.1. Total erythrocyte count (TEC)
In rabbits of group B, treated with arsenic trioxide @100ppm, TEC started to decrease from 20 days onward and the significant reduction (P<0.05) was observed on 40 days of feeding.The decrease was highly significant (P<0.01) on 60 days of feeding (Table -2).The results was in accordance with the findings of few workers (Chinoy et al., 2004;Rana et al., 2008;Ferzand et al., 2012;Gyasi et al., 2012).The suppressed TEC, Hb concentration could be due to impaired function of bone marrow and a suppression of erythropoiesis following chronic arsenic toxicity.In other groups, TEC values were slightly altered but not statistically significant.This might be due to interaction of tannic acid, DSHP, Alum with arsenic trioxide and the use of SCIF bed filtered water.It was noticed that arsenic toxicity depends on the nature of arsenic compounds and roots of absorption.High concentration of arsine (10 ppm) caused death within hours due to red blood cell haemolysis in man.Low levels of arsenic (0.5-5.0 ppm) brought about these effects in a few weeks, and an average concentration of 0.5 mg/l (0.2mg/m3) was considered acceptable in the work place for human (Sittig, 1985).Renal damage was secondary and occured due to clogging of nephrons with hemolytic debris.Mono-, di-, and trimethylarsines were strong irritants but were found less hemolytic than arsine (NIOSH, 1979).Arsine exposure by humans was found fatal without proper therapy.Arsine breaks down in the body to inorganic arsenic and methylated derivatives which was less toxic than arsine (Fowler and Weissburg, 1974).The mechanism of hemolysis involved depletion of intracellular GSH, resulting in oxidation of sulfhydryl groups in the hemoglobin from ferrous to ferric in mice and rats.Haemocyanin combines with arsenic, which reduced oxygen uptake by cells and therapy prevents hatching (Calabrese et al., 1987.Table 1.Effect of arsenic trioxide along with tannic acid, DSHP, alum and effects of SCIF-bed filtrated water along with alum respectively on body weight (gm) and in rabbits.Table 2. Effect of arsenic trioxide along with tannic acid, DSHP, alum and effects of SCIF-bed filtrated water along with alum respectively on total erythrocyte count(TEC)(X 10 6 /mm 3 ) and total leukocyte count(TLC)( X 10 6 /mm 3 )in rabbits.

Total leukocyte count (TLC)
In rabbits of group B, treated with arsenic trioxide @100ppm, the TLC count was increased from day 20, from day 40 it was significant (P<0.05) and from day 60 it was highly significant (P<0.01)(Table -2).The results was in accordance with the findings of yeasmin et al., (2011) andSarker et al., (2012) in mice but dissimilar to the findings of Ferzand et al., (2012); Gyasi et al., 2012 in mice.This might be due to counteract the poisonous effect of arsenic, duration of exposure and dose variability.The haematopoietic system was also affected by both short-and long-term arsenic exposures.Anemia and leukopenia were common effects of poisoning and have been reported as resulting from acute, intermediate and chronic oral exposures (Armstrong et al., 1984).These effects might be due to a direct haemolytic or cytotoxic effect on the blood cells (Lerman et al., 1980).In other groups, TEC values were not altered significantly.This might be due to interaction of tannic acid, DSHP, Alum with arsenic trioxide and the use of SCIF bed filtered water.

Hemoglobin content (Hb content)
In rabbits of group B, treated with arsenic trioxide @100ppm, hemoglobin content started to reduce from day 20 onward and on days 40 amount decreased was significant (P<0.05).However, highly significant (P<0.01)change was observed at 60 days of feeding (Table -3).The results was in accordance with the findings of Blair et al., (1990);Ferzand et al., (2012); Gyasi et al., (2012) andSarker et al., (2012).In the same type of studies conducted by Breton et al., (2006) and Gupta and Flora (2006) and they examined the hematological alteration due to arsenic and their results were correlated with our results that TEC and Hb was decreased.This could be due to binding ability of arsenic to Hb that led to inhibition of heme synthesis pathway.PCV decreasement was the result of the decreasement of prior parameters.In other groups, hemoglobin content was slightly altered but not statistically significant.This might be due to interaction of tannic acid, DSHP, Alum with arsenic trioxide and the use of SCIF bed filtered water.

Packed cell volume (PCV)
In rabbits of group B, treated with arsenic trioxide @100ppm, the PCV value started to decrease from day 20 onward, from day 40 reduction was significant (P<0.05) and from day 60 it was highly significant (P<0.01).
The result was in accordance with the findings of Islam et al., (2011).The decrease in PCV could be due to reduction of red marrow in chronically exposed animals.In other groups, the PCV values were not altered significantly (Table -3).

Erythrocyte sedimentation rate (ESR)
In rabbits of group B, treated with arsenic trioxide @100ppm, the ESR values was increased from day 20, significant (P<0.05)increased from day 40 and from day 60 it was highly significant (P<0.01).The result was in accordance with the findings of Islam et al., (2011).This increase ESR value might be due to the alterations of the other parameters of blood.In other groups, TEC values were not altered significantly (Table -3).

Conclusions
Treatment with arsenic oxide at low doses has harmful effects on experimental animals including hematological alterations.Therefore, intake of alum treated SCIF bed filtered water might be helpful to reduce the body burden of arsenic toxicities.