Effects of dengue virus infection on peripheral blood cells of mice exposed to hexavalent chromium with drinking water
Background & objectives: The occupational and non-occupational exposure to hexavaient chromium Cr (VI) is common. The effect of chromium compromises the immune response of the host. Dengue virus (DV) infection causes various changes in the peripheral blood cells. It is, therefore, possible that the chromium toxicity may affect the disease process during DV infection. The present study aims to study the effects of dengue virus infection on peripheral blood cells of mice fed Cr (VI) with drinking water.
Methods: One group of mice was given ad libitum drinking water containing Cr (VI) and the other group used as the normal control mice was given plain water to drink. At the 3, 6 and 9 wk of Cr (VI) drinking, a set of mice from each group was inoculated intracerebrally (ic) with DV and studied at the 4th and 8th day post inoculation.
Results: It was observed that Cr (VI) drinking led to reduction in lymphocytes, haemoglobin and the haematocrit values while the granulocyte, monocyte and platelet counts were increased. On the other hand, most of the parameters were decreased following inoculation of normal mice with DV. In Cr (VI)-fed mice the effects of DV infection were minimal. The most significant finding of these experiments was that the reduction in platelet counts following inoculation with DV was markedly less in Cr (VI)-fed mice than that in DV-inoculated normal control mice.
Interpretation & conclusion: Cr(VI) compounds have been declared as a potent occupational carcinogen. On the contrary, Cr(III) salts such as chromium polynicotinate, chromium chloride and chromium picolinate, are used as micronutrients and nutritional supplements, and have been shown to exhibit health benefits in animals and humans. Whether therapeutic doses of chromium (III) compounds may be able to prevent the DV-induced fall in platelet counts, needs to be investigated.
Key words Blood cells – Cr (VI) – dengue virus – leucocytes – lymphocyte – monocyte – platelet – polymorphonuclear cells – RBC
Hexavalent chromium [Cr (VI)] occurs naturally in the environment and is widely used in paints, metal finishes, steel including stainless steel manufacturing, alloy cast irons, chrome and wood treatment and leather tanning. Cr (VI) ingested with food such as vegetables, meat, etc., is reduced to Cr (III) before entering the blood stream. Cr (VI) is highly toxic to all forms of living organisms1-3. Chromium enters the body through the lungs, gastrointestinal tract, and to a lower extent through skin4. Non-occupational exposure occurs via ingestion of chromium containing food and water5. Regardless of route of exposure, Cr(III) is poorly absorbed whereas Cr(VI) is more readily absorbed4. Cr (VI) compounds that enter the bloodstream are transported into red blood cells (RBCs) via sulphate anion channels6. Cr(VI) is rapidly reduced to Cr(III) via unstable intermediates [Cr(V) and Cr(IV)] inside the RBC. The reduced product binds to haemoglobin and other intracellular proteins, resulting in elevation of total chromium levels in the RBC fraction of blood for several weeks6-8. The main routes for the excretion of chromium are via kidney/ urine and the bile/feces5,9.
During dengue virus (DV) infection various changes occur in the peripheral blood cells. The total leucocyte count in patients with dengue infection varies from mild leucopaeniato moderate leucocytosis with predominance of lymphocytes. The haematocrit value increases during the first three days but gradually decreases from day four to nine. The platelet count is usually normal during the first three days. Thrombocytopaenia in more than 80 per cent cases begins during febrile stage and reaches its lowest value during the haemorrhagic phase (dengue haemorrhagic fever, DHF) of illness10. Thrombocytopaenia is one of the simple diagnostic criteria proposed by World Health Organization (WHO) for diagnosis of DHF11. Constant haematological abnormalities occurring in DHF include bone marrow suppression, leucopaenia and thrombocytopaenia12. DV does not produce any clinical illness in any animal species and there is no animal model similar to human DHF. In absence of such a model, inoculated mice have been used extensively to answer several questions related to dengue infection13-18.
DV infection is endemic all over India so is the occupational and non-occupational exposure to Cr (VI). The effect of chromium compromises the immune response of the host20. It is, therefore, possible that the chromium toxicity may affect the disease process during DV infection. The present experiments were, therefore, conducted to study the effects of dengue virus infection on peripheral blood cells of mice fed Cr (VI) with drinking water.
Material & Methods
Animals: The study was carried out on Swiss mice weighing 25-30 g, aged 6-8 wk obtained from the animal breeding facility of the Industrial Toxicology Research Centre, Lucknow. Clearance from the Animal Ethical Commettee of the Institute was obtained for the use of animals. Mice were maintained on pellet diet.
Plan of study: One group of mice (n=72) were given ad libitum drinking water containing Cr (VI) and the second group (n=24) was given plain water to drink (Fig.1). At the 3, 6 and 9 wk of Cr (VI) drinking, a group of mice (12 animals in each group) were inoculated ic with dengue virus in doses of 1000 LD^sub 50^13. The mice were killed in groups at days 4 (6 animals) and 8 (6 animals) post virus inoculation (pi) and various investigations were done.
Chromium treatment of mice: Each group with 6 mice was given ad libitum drinking water containing 250 ppm chromium (VI) in form of potassium dichromate for 3, 6 and 9 wk. Oral LD^sub 50^ values for Cr(VI) compounds was 300 mg/kg for potassium chromate in the mouse19. From the daily consumption of water it was found that the average daily intake of Cr (VI) per mouse was 14.8 mg/kg.
Dengue virus: Dengue type 2 virus (DV), strain P23085 (kindly supplied by the Director, National Institute of Virology, Pune) and was used in the form of infected infant mouse brain suspension13. Mice were inoculated with 1000 LD^sub 50^ of DV intracerebrally (ic) in doses of 0.03 ml.
Collection of blood sample: Animals were sacrificed and blood samples were collected from the jugular vein in tubes containing 0.5 ml freshly prepared 10 per cent K^sub 3^ EDTA solution as anticoagulant. The samples were shaken gently.
Haematologicalstudy: The following estimations were performed immediately with the help of a fully automatic haematology analyzer (Bayer; Technicon H1*E, USA): total leucocyte count (TLC), differential leucocyte count (DLC), total RBC count, haematocrit (Hct), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and platelet counts.
Statistical analysis: Two-way analysis of variance (Two-way ANOVA) was done to compare the mean values of the outcome variables (TLC, DLC, RBC count, Hb, Hct, MCV, MCHC, and platelet count) considering chromium exposure and dengue virus infection to animals as independent variables. Prior to this homogeneity of variance between the treated groups were ascertained. The two-group comparison was done by calculating least significant difference at 5 per cent level of significance.
Effects of dengue virus inoculation on mice: The mice remained apparently healthy up to the day 4 of DV inoculation. The back was arched and the fur was ruffled from day 5 pi. By the 8th day the mice became seriously ill with the development of hind limb paralysis and all of them died by the day 10 pi. Therefore, the experiments were conducted on days 4 and 8 post inoculation of the dengue virus. The clinical dengue illness in Cr (VI)-fed mice was similar to that in normal mice.
Effects on leucocytes: In normal control mice the total leucocyte count was 7±0.9×10^sup 3^/µl with 83 per cent lymphocytes and 11 per cent granulocytes. By giving Cr(VI) in drinking water for 3 wk the total count was reduced significantly (P
Effects on red blood cells: The total red blood cell (RBC) count in normal mice was 8.3 ± 0.3 µl. By feeding Cr(VI) the red blood cell count increased significantly (P
Effects on platelets: The platelet count in the blood of normal control mice was 370 ± 15×10^sup 6^/µl with Cr(VI) drinking the total platelet count was significantly increased to 622 ±128 x10^sup 6^/µl (P
When normal mice were inoculated with DV slight change was observed in platelet distribution width and the mean platelet volume (Table III). Inoculation of dengue virus in Cr(VI) treated mice resulted in the decrease of mean platelet volume (P
The findings of the present study showed a marked reduction in lymphocyte percentage and increase in the granulocyte, monocyte and platelet counts in mice fed Cr(VI) with drinking water. Differential leucocyte counts revealed that percentage of lymphocytes decreased by 50 per cent while the percentage of polymorphonuclears and monocytes was increased gradually. Similar findings have been reported in fish exposed to chromium21. Rats exposed to atmosphere containing soluble potassium chromate have significantly increased levels of polymorphonuclear cells and monocytes22. A significant increase in polymorphonuclear cells has been reported in a 25 yr old women who drank a solution containing potassium dichromate23. The normal mice inoculated with DV showed a 48 per cent decrease in total leucocyte count. The number of polymorphonuclears was also reduced in the present study. Patients with dengue fever have mild leucopaenia to mild leucocytosis associated with lymphocytosis11,24. The interesting finding in the present study was that Cr(VI)-fed mice inoculated with DV showed marked reduction in number of lymphocytes, while the number of polymorphonuclears was not affected significantly.
At three week of Cr(VI) drinking the red blood cell count was increased by 29 per cent while at 6th and 9th wk it remained the same in our study. Glaser et al25,26 reported that rats exposed to Cr(VI) for 18 months had increased red blood cell counts. A reduction in MCV and MCHC was observed at the end of 3rd wk of Cr(VI) drinking. Rats and mice fed with potassium dichromate showed slightly reduced MCV and MCH values at earlier periods and low doses of Cr (VI) but the decrease was marked with the higher doses given for longer periods27-29. In the present study, it was observed that after 9 wk of Cr(VI) drinking haematocrit and haemoglobin contents were decreased. The decrease in haemoglobin appears to be due to inhibition of its biosynthesis. In the initial stage of pathway, succinyl Co-A combines with glycine to form the first of the intermediates, δ-amino levulinic acid. Chromium may cause an increase in SDH synthesis and therefore decrease the succinyl Co-A pool. It may also interact with serine, which is the precursor of glycine and may thus decrease the serine pool. Chromium causes the DNA damage, producing inhibition of the activities of one or more enzymes involved in heme synthesis30,31. Sharma et al32 have reported that in an 18 yr old women who ingested few grams of potassium dichromate showed decreased haemoglobin and haematocrit on day 4 after ingestion. In another study, a 44 yr old man had decreased haemoglobin levels 9 days after ingestion of chromium33 .
Haemoglobin concentration was decreased by 42 per cent on day 8 in normal mice inoculated with DV. The haematocrit value was also decreased by 40 per cent. Pimpan and Prasert11 reported that in patients with DHF haematocrit value increased initially but later on it decreased. Gastrointestinal bleeding might initially be occult and usually manifested as a drop in haematocrit without clinical improvement34,35. Cr(VI) feeding led to an increase in platelet counts in the present study, while in normal mice inoculated with DV, platelet count was decreased significantly at days 4 and 8 post inoculation. Thrombocytopenia is one of the simple diagnostic criteria proposed by the WHO for clinical diagnosis of DHF11. The cause of thrombocytopenia is either impaired megakaryocytes production or increased platelet distruction10.
Cr(VI) compounds have been declared as a potent occupational carcinogen among workers in chrome plating, stainless steel, and pigment industries3. The cells of immune system have capacity to reduce toxic Cr (VI) to non-toxic Cr (III) and in the process some immune functions are disturbed20,36. On the contrary, Cr (III) salts such as chromium polynicotinate, chromium chloride and chromium picolinate, are used as micronutrients and nutritional supplements, and have been demonstrated to exhibit significant health benefits in animals and humans9,37,38.
In conclusion, the reduction in platelet counts following inoculation with DV was markedly less in Cr (VI)-fed mice than that seen in DV-inoculated normal control mice. Further studies need to be done to see whether Cr (III) therapy with chromium polynicotinate, chromium chloride or chromium picolinate will be able to prevent the pathognomonic fall in platelet count in patients with dengue virus infection.
The financial support received by the first author (RS) in form of Senior Research Fellow from the Indian Council of Medical Research, New Delhi is acknowledged. The authors thank Dr Neeraj Mathur for the help in statistical analysis.
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Richa Shrivastava+, S. Srivastava*, R.K. Upreti & U.C. Chaturvedi
Industrial Toxicology Research Centre & *Central Drug Research Institute, Lucknow, India
Received September 23, 2004
+ Present address: Central Food Technology Research Institute, Mysore
Reprint requests’. Prof. U.C. Chaturvedi, 201-Annapurna Apartments, No.1, Bishop Rocky Street
Faizabad Road, Lucknow 226007, India
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