Evaluation of the Haematological Responses to High Nitrate Exposure in RabbitsCorrespondence Address :
Dr. Manoj Kumar Sharma
III/5, Staff quarters, Medical college campus,
Jhalawar Medical college, Jhalawar - 326001 (Rajasthan)
Phone: 07432-233412; Mob. 09667334455; 09929391333
Introduction: Nitrate is a common pollutant in the drinking water of Rajasthan as well as in the rest of India. The maximum permissible limit for the nitrate ion in drinking water has been set at 50 mg/l by the WHO and at 45 mg/ l by the Bureau of Indian Standards. In the body, nitrate is reduced to nitrite and the absorption of nitrite leads to methemoglobinaemia. Nitrate toxicity has been observed frequently in ruminants which has been fed on a variety of plant materials which had high nitrate content.
Aim: The present study examined a possible correlation between the nitrate concentration in drinking water and the blood profile changes in rabbits.
Materials and Methods: Rabbits were selected for the study because the pH of their stomachs was similar to that of human beings. We included 5 groups of rabbits, with 2 rabbits in each group. The groups were named A, B, C, D and E respectively. The nitrate concentrations in the drinking water were 45 mg/l, 100 mg/ l,200 mg / l,400 mg / l and 500 mg/ l in the groups A to E respectively. The group A was considered as the control group. After 120 days of the ingestion of sodium nitrate in drinking water, blood samples were collected from the rabbits of each group and were observed.
Results and Conclusion: The maximum haematological parameters such as WBC, lymphocytes, MCHC, RBC, Hb and basophills showed decreased values in the higher groups as compared to the values of the control group. The PCV and MCV showed increased values in the higher groups as compared to the values of the control group. The present study indicated that the symptoms of nitrate toxicity were associated with the low oxygen carrying capacity of blood in the affected animals. Nitrate toxicity is a potential health hazard and the scarcity of data on the nitrate toxicity in blood created an interest in attempting the present study.
Nitrate, Nitrite, Blood, Methemoglobin, Toxicity, Rabbits
A major part of the Indian population is exposed to nitrate through dietary sources (1). The maximum permissible limit for the nitrate ion in drinking water has been set at 50 mg/l by the WHO and at 45 mg/l by the Bureau of Indian Standards (2), (3), (4). In several countries, especially in India, the consumption of water which contains high nitrate concentrations, sometimes as high as 500 mg/l, is common (4),(5),(6). In the human body, nitrate is reduced to nitrites, which cause methemoglobinaemia (6). When nitrate is ingested it is reduced to nitrites in the intestinal tract, although some studies suggested that this process may start in the oral cavity (6)-,7]. The nitrite ions are absorbed in to the blood stream where they convert haemoglobin to the methemoglobin (7). The health risk from the exposure to nitrate is therefore related not only to its concentration in drinking water and food, but also to a condition which is conducive to its reduction to nitrites (8). This action may be brought about in one of the following ways (9): By the direct action of the oxidant or by the action of a hydrogen donor in the presence of oxygen, or by auto oxidation. In the presence of nitrites, the ferrous ion of haemoglobin gets directly oxidized to the ferric state. Normally, methemoglobin is formed by the following reaction:
Hb+3 +Red.Cyt b5 ----------> Hb+2 + Cyt b5
Reduced cytochrome b5 is generated by the enzyme, cyt.b5 reductase: Original Article Research Section
Oxy cytb5 + NADH ----------> Red cyt b5 + NAD.
Thus, the enzyme, cyt b5 reductase plays a vital role in counter acting the effect of nitrate ingestion. We chose rabbits for the study because the pH of their stomachs is similar to that of human beings (6). So, it was proposed to study the effects of nitrate toxicity in the blood profile of rabbits .
This study was conducted on five groups of rabbits, with 2 rabbits in each group. The rabbits were used for the study because the pH of their stomachs was similar to that of infants (pH= 3.0-5.0) (6). The rabbits were three and a half to four months old and their weights varied from 1.310 kg to 1.720 kg. The 5 groups were identified as A, B, C, D and E. Ad libitum quantity of water which contained 45, 100, 200, 400 and 500 mg/liter nitrate (in form of NaNO3) and food which was soaked in the same water were given to the groups A, B, C, D and E respectively. The group which consumed 45mg/ liter of nitrate served as the control group. After the completion of the experimental period, the blood samples were collected from the rabbits of each group in heparinized vials according to the guidelines of ICMR and they were sent to the central lab of Swai Man Singh Hospital and Medical College, Jaipur for haematological estimation. The results of the blood estimation were recorded and shown by using an autoanalyzer (Table/Fig 3),(Table/Fig 4),(Table/Fig 5), (Table/Fig 6),(Table/Fig 7).
The values of the blood for all the groups were obtained by using an autoanalyzer at the Swai Man Singh Hospital , Jaipur and they were compared with the values of the blood of group A (control group), separately one by one (Table/Fig 1),(Table/Fig 2). The WBC and MCHC showed increased values in the blood of group B only and continuous decreased values in that of group C, D and E respectively as compared to the values of the blood of group A. The lymphocytes and monocytes showed increased values in the blood of groups C and D, but in group E, decreased values was observed as compared to the values in the blood of group A. The basophils showed increased values in the blood of groups B and C and decreased values in the blood of groups D and E as compared to the values of the blood of group A respectively. RBC and Hb showed decreased values in the blood of groups B, D and E as compared to the values of the control group, but PCV showed decreased values in blood of group B only as compared to the values of the blood of group A. MCV showed increased values in the blood of the rabbits of all the groups.MCH showed decreased values only in the blood of group C and increased values in the blood of groups B and D respectively, but group E showed the same value as was observed in the blood of group A.
The presence of high nitrate concentrations in drinking water causes severe methemoglobinaemia and clinical cyanosis in infantsand adults. The present study indicates that the excess intake of nitrate adversely affects the blood profile in rabbits. Marshall W et al (9) observed the action of nitrites on blood. The interaction of sodium nitrite and blood takes place in three stages: (a) An induction period (b) A reactionary period (c) A terminal period which is often prolonged, during which the products of the reaction, chiefly methemoglobin, pass into haematin and other degradation products. Darling and Roughton (10) described that the action of nitrite on haemoglobin was extremely complicated. It varies with the molecular ratio of nitrite to haemoglobin, pH, the presence or absence of O2 and reducing agents and possibly with other factors.
In the present study, Hb showed increased values in the blood of group C as compared to the values of the control group. This finding was consistent with the findings of Ogur R et al (11) . The WBC showed decreased values in groups C, D and E (in the higher concentration group) as compared to the values of the controls. This finding was consistent with the findings of Tepperman J et al (12). RBCs showed decreased values in the higher groups as in the groups D and E. These findings were in accordance with the results of Bodannsky O et al (13) who also reported that the repeated administration of methemoglobin producing agents like nitrate, acetanilide ,acetophanetidine, etc. may cause haemolytic anaemia but that it may be dose dependant.
MCV showed continuous increased values in the higher groups as in the groups A, B, C, D and E in the present study, which showed similarity with the findings of Barry N. Madison et al (14) who studied the haematological responses of acute nitrite exposure in fishes. They observed that nitrite became more lethal in the presence of high bacterial activity due to the waste build up in the body. Van Loon et al (15) found that the chronic administration of acetanilide (up to 36 mg/kg) caused little or no haematological changes in dogs, but that larger doses produced haemolytic anaemia.
Nitrates are potential health hazards and they have been known to cause methemoglobinaemia and other changes in the blood profile (16). The scarcity of data on the nitrate toxicity in blood created an interest in attempting the present study, but a more elaborative study with larger number of samples is required to establish a positive correlation.
ABBREVIATIONS 1. Cyt b5 Cytochrome b5 2. Hb+2 Ferrous state of hemoglobin 3. Hb+3 Ferric state of hemoglobin 4. ICMR Indian Council of Medical Research 5. Kg Kilogram 6. mg Milligram 7. NAD Nicotinamide adenine dinucleotide 8. NADH Nicotinamide adenine dinucleotide hydrogenase 9. pH Negative logarithm of hydrogen ion 10. Red cyt b5 Rduced cytochrome b5 11. Oxy cytb5 Oxidized cytochrome b5 12. WHO World Health Organization 13. WBC White Blood Cells14. RBC Red Blood Cells 15. Hb Hemoglobin 16. PCV Packed Cell Volume 17. MCV Mean Cell Volume 18. MCH Mean Cell Hemoglobin 19. MCHC Mean Cell Hemoglobin Concentration. 20. N% Neutrophills 21. L% Lymphocytes 22. M% Monocytes 23. B% Basophills.
I am extremely thankful to my esteemed teacher ,distinguished guide and true mentor Dr. (Mrs.) Neelam Bapna, Ex professor and Head, Department of Anatomy, Swai Man Singh Medical College, Jaipur, India for the precise guidance, valuable suggestions and constant encouragement without which it would not have been possible for me to start and finish this research work. I am also thankful to Dr. Suresh Joshi, Ex Deputy Director, University Grant Commission Academic staff college and Associate Professor, Department of Zoology, University of Rajasthan, Jaipur, India whose initiative gave me a sense of professional duty and pride. I am equally thankful to Dr. Dhindsa , Superintendent Chemist, Public Health and Engineering Department for his cooperation in the estimation of nitrate in drinking water and other chemical evaluations during my research work. Mrs. Hemlata sharma, Lecturer, Department of Anatomy, Jhalawar Medical College, Jhalawar, India was helpful in preparation of this manuscript and helped me in quite a lot with the leg work. Equally important was the friendly guidance and cooperation of Dr. Vijay Shekhawat, Animal Surgeon and Mr. Manohar singh, Animal House attendant, Swai Man Singh Medical College, Jaipur, India for their help in providing a healthy environment for the rabbits during my experiments. The whole research work was funded by myself only.
FINANCIAL OR OTHER COMPETING INTERESTS: NONE.
Date of Submission: Aug 28, 2011
Date of Peer Review: Oct 21, 2011
Date of Acceptance: Dec 20, 2011
Date of Publishing: Feb 15, 2012
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