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Liver phosphatases in mice treated with lead during murine ancylostomiasis
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 61 (2005) 134–136
Liver phosphatases in mice treated with lead during murine ancylostomiasis B.D.J. Satyalatha and V.V. Vardhani* Department of Zoology, Nagarjuna University, Nagarjuna Nagar (A.P.) 522 510, India Received 17 October 2002; received in revised form 12 December 2003; accepted 27 January 2004
Abstract Both infective larvae and pollutants disturb the homeostasis of female Swiss albino mice, causing ancylostomiasis and severe immunophysiological changes. In our experimental design, mice in group A were fed 0.01 mg of lead nitrate before infection with 500 larvae of Ancylostoma caninum, mice in group B were infected with larvae without previous lead treatment, and group C received lead nitrate treatment alone. Control animals in group D were neither treated with lead nitrate nor infected with larvae. While all three experimental groups showed disturbances in liver alkaline phosphatase (ALP) and acid phosphatase (ACP), group C receiving lead nitrate treatment alone and group A receiving lead nitrate with infection showed increased levels of ACP and decreased levels of ALP. r 2004 Elsevier Inc. All rights reserved. Keywords: Mice; Ancylostoma caninum; Liver; Phosphatases
1. Introduction Hookworm infection has long been known as one of the major cosmopolitan diseases of humanity. The zoonosis caused by canine hookworms is an interesting aspect to study because of the close association between humans and dogs (Lynden et al., 1998). Hookworm larvae penetrate the unbroken skin of humans and migrate through subcutaneous tissue. Since humans are not the natural hosts, the infective larvae are unable to complete their migratory cycle and produce skin lesions (Gilles, 1985). Human infection occurs in areas of recreational exposure to contaminated soil (Matgor et al., 1996). The effect of essential minerals and heavy metals (Co, Cu, Fe, Zn) and Ancylostoma caninum infection on the humoral and cellular components of immune response has been studied (Gowri and Vardhani, 1992; Ahmad et al., 1994; Boroskova et al., 1994). Mice exposed to oral doses of lead and challenged with a single dose of Hymenolepis nana eggs harbored significantly greater worm burden than did controls (Agrawal et al., 1989). The effect of heavy metals and/ or A. caninum infection on biochemical components of *Corresponding author. Fax: +91-863-2293378. E-mail address: [email protected] (V.V. Vardhani). 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.01.013
various experimental animals has been studied (Vardhani, 1986; Trehan and Maneesha, 1991; Nirmala, 1999; Lomte et al., 2000). In the case of humans, the contamination of air, food, and water (tropical diseases) led to ‘‘occupational diseases’’ caused by overexposure to a pollutant by virtue of an individual’s occupation. Some of the important contributors to environmental degradation are industrial effluents and automobile exhausts. Lead poisoning may occur in children and/or adults as a result of accidental or occupational ingestion. Lead is absorbed through the gastrointestinal tract or lungs; the absorbed lead is distributed into the liver, kidneys, and bone marrow, causing a direct toxic effect in these organs. Therefore, the present work was planned to study the effect of lead nitrate on liver phosphatases of female Swiss albino mice after oral treatment and/or infection.
2. Materials and methods Infective A. caninum larvae were cultured following the method of Sen et al. (1965). Mice in group A were treated orally with lead nitrate for 4 days (0.01 mg/ mouse) and infected with an oral dose of 500 larvae of
ARTICLE IN PRESS B.D.J. Satyalatha, V.V. Vardhani / Ecotoxicology and Environmental Safety 61 (2005) 134–136
135
Table 1 Liver enzymes, acid phosphatase, alkaline phosphatase values, and worm load in experimental mice at different times after infection. Day of necropsy
Experimental groups A
1
4
9
16
30
B
C
ACP (IU/L)
ALP (IU/L)
WL
ACP (IU/L)
ALP (IU/L)
WL
ACP (IU/L)
ALP (IU/L)
17.778.8 t ¼ 8:02 Po0:05 22.178.7 8.7 t ¼ 9:98 Po0:05 14.678.9 t ¼ 5:02 Po0:05 12.778.7 t ¼ 3:35 Po0:05 11. 378.8 t ¼ 1:70 P40:05
131.47159.7 t ¼ 2:02 P40:05 149.17159.6 t ¼ 0:68 P40:05 159.97159.7 t ¼ 0:10 P40:05 165.77159.8 t ¼ 3:62 P40:05 175.37159, 7 t ¼ 5:12 Po0:05
56 (11.2%)
15.278.8 t ¼ 5:02 Po0:05 14.378.7 t ¼ 4:90 Po0:05 11.878.9 t ¼ 1:89 P40:05 10.778.7 t ¼ 1:04 P40:05 9.678.8 t ¼ 0:98 P40:05
158.77159.7 t ¼ 0:02 P40:05 158.57159.7 t ¼ 0:96 P40:05 157.57159. 7 t ¼ 1:02 P40:05 157.47159.7 t ¼ 2:01 P40:05 156.97159.7 t ¼ 2:96 P40:05
58.0 (11.6%)
16.678.8 t ¼ 6:04 Po0:05 15.278.7 t ¼ 3:75 Po0:05 13.078.9 t ¼ 5:02 Po0:05 12.078.7 t ¼ 2:02 P40:05 11.078.8 t ¼ 1:96 P40:05
145.0 7159.7 t ¼ 2:08 Po0:05 1307159.6 t ¼ 2:20 Po0:05 1267159.7 t ¼ 2:14 Po0:05 124.67159.8 t ¼ 2:13 Po0:05 120.27159.7 t ¼ 2:24 Po0:05
50 (10.0%)
–
–
–
0.2 (0.4%)
–
–
–
Abbreviations: ACP, acid phosphatase; ALP, alkaline phosphatase; WL, worm load. Values are expressed as means derived from five observations.
A. caninum on day 5 (n ¼ 25), group B (n ¼ 25) with an equal amount of saline for 4 days and infected with an oral dose of 500 larvae on day 5, and group C (n ¼ 25) with lead nitrate alone (0.01 mg/mouse) for 4 days. Mice in group D were kept as untreated and uninfected controls. Five mice from each experimental group were sacrificed on day 1, 4, 9, 16, and 30 after infection (groups A and B) and lead nitrate treatment (group C) for the collection of larvae (from groups A and B) and for enzymatic assays from liver. The control mice were sacrificed for enzymatic assays on the same days as just described. The livers were excised and analyzed for phosphatases by the method described by Jaffee and Badansky (1943). Worm burden from liver was estimated from larval recoveries by Baermann’s technique.
3. Results and discussion The mean values of liver phosphatases from the experimental groups (A–C) and the control (group D) animals and worm load (groups A and B) for the 30-day experimental period with their statistical values are shown in Table 1. In group A mice (treated+infected), there was a considerable rise in acid phosphatase (ACP) from day 1 to 30, with a peak response on day 4 when compared with uninfected and untreated controls (group D); ACP levels then decreased gradually from that peak, reaching an almost normal value. The migration of larvae into the liver might have caused pathological reactions resulting in the overexpression of ACP. The increase was significant only when compared with controls (Po0:05). The amount of alkaline
phosphatase (ALP) in group A mice was below normal from day 1 to 16 but had significantly increased by day 30. This increase was found to be significant (Po0:05) in comparison with controls and with mice receiving the lead nitrate alone (group C), but not with mice infected with 500 larvae (group B). It is interesting to note that on day 30, the ACP value that had been high on day 1 decreased to a normal value, while the ALP value that had been below normal on day 1 increased significantly. In group B mice (untreated+infected), ACP values increased (Po0:05) from day 1 to 4 in comparison with controls, but not with mice receiving lead nitrate alone (group C); in these mice there was a gradual decrease of ACP from day 1 to 30. ALP activity was reduced slightly (P40:05) throughout the experimental period when compared with group D (there was a decrease in comparison with group C of below normal values). The activity of ALP remained almost stable during day 1 to 30. In group C mice receiving only lead, there was a twofold (Po0:05) rise in ACP from day 1 to 9 in comparison with controls; the ACP activity was not significant in comparison with groups A (treated+infected) and B (infected only). The increased amount of ACP (on day 1) decreased afterwards day by day, and the value on day 30 (11.0 units) was almost equal to that of group A (11.3 units). There was a below normal value (Po0:05) of ALP from day 1 to day 30 in comparison with controls and both experimental groups A and B. The decreased value of ALP on day 1 (in comparison with the control group) continued to decrease by B15% by day 30.
ARTICLE IN PRESS 136
B.D.J. Satyalatha, V.V. Vardhani / Ecotoxicology and Environmental Safety 61 (2005) 134–136
4. Statistical analysis ACP activities varied significantly in all the experimental groups when compared with controls. In group A, the values of ACP increased significantly from day 1 to 16 when compared with controls; from day 4, the ACP amount decreased gradually, reaching an almost nonsignificant level by day 30. Conversely, the values of ALP decreased to below normal levels from day 1 to 16 and increased significantly by day 30. In group B, ACP increased significantly on day 1 and 4, and there was a gradual decline from day 4 to day 30, reaching normal values by day 30. The retention of larval burden, increase of ACP on day 1 and 4, and the gradual decrease from day 4 to day 30 suggest an equivalent immune response in groups A and B. The decreased (group A) and stable (group B) nonsignificant values of ALP (except the significant value of group A on day 30) indicate that the hookworm infection may not alter the level of this enzyme. The nonsignificant values of ACP (except on days 1 and 4) and of ALP of group C suggest that a dose of 0.01 mg of lead nitrate may not bring about significant alterations in the levels of liver phosphatases. The larval burden in groups A and B suggests that adverse interactions took place between the host and lead and/or infective larvae, which in turn triggered a nonspecific reaction that disturbed the level of the liver enzymes. The results are comparable to Agrawal et al. (1989), who found increased worm burden in H. nana-infected mice exposed to low oral doses of lead. Increased larval burden is related to reduced immunity due to decreased globulin production (Griggs, 1964). The increased values of ALP (only from day 1 to 9 in group A) in experimental groups from 1 to 30 days of the experimental period indicate a correlation with the increased value of ACP. The increased value of ACP and decreased value of ALP suggest the role of lead and/ or infection. Lead may inhibit the enzyme d-aminolevulinic acid dehydrate, which is the most sensitive enzyme in the pathway, as suggested by Griggs (1964). Either a small dose of lead along with infection or lead alone resulted in decreased values of ALP and increased values of ACP in mice.
5. Conclusions The alteration of liver phosphatases (ACP and ALP) in mice treated with lead is comparable to that of Karthikeyan (2001), who also found cholestasis of the liver in rats treated with endosulfan. Our results indicate that the lead treatment alone and in combination with
hookworm infection induces toxic reactions in the liver, with subsequent disturbances in the metabolic–enzymatic homeostasis.
Acknowledgments We are thankful to Lutheran World Federation, Geneva for providing funds to one of us (B.D.J. Satyalatha) to carry out this work.
References Agrawal, R., Chowdary, S., Johri, G.N., 1989. Effect of lead on the retention of parasitic burden and certain hematological changes in the Swiss albino mice, Mus musculus albinos. Proc. Natl. Acad. Sci. USA 59, 33–37. Ahmad, S., Singh, V., Rao, G.S., 1994. Antioxidant potential in serum and liver of albino rats exposed to benzene. Indian J. Exp. Biol. 32, 203–206. Boroskova, Z., Benkova, M., Soltys, J., Krupicer, I., 1994. Effect of heavy metal emission on the immune response of sheep with experimental fasciolosis. Parasite Immunol. 16, 389–391. Gilles, H.M., 1985. Selective primary health care strategies for control of disease in the developing world. XVII. Hookworm infection and anaemia. Rev. Infect. Dis. 7, 111–118. Gowri, P., Vardhani, V.V., 1992. Eosinophilia in murine ancylostomiasis. J. Hyg. Epidemiol. Microbiol. Immunol. 36, 175–180. Griggs, R.C., 1964. Lead poisoning: hematological aspects. In: Progress in Hematology, Grune and Stratton, New York, p. 117. Jaffee, H.C., Badansky, O., 1943. Diagnostic significance of serum alkaline and acid phosphatase values in relation to bone disease. Bull. NY Acad. Med. 19, 831–848. Karthikeyan, S., 2001. Effect of chronic administration of endosulfan on transaminases, alkaline phosphatase value in relation to bone disease. J. Ecotoxicol. Environ. Monit. 1 (3), 169–172. Lomte, V.S., Rajendra, T.C., Sultana, M., 2000. Changes in protein content of fresh water Bivalve, Parreysia cylindrica on exposure to nickel chloride and lead chloride. Indian J. Ecoenviron. Ecoplanning 3, 63–65. Lynden, L.M., Tandorc, V., Yadav, A.K., 1998. Hookworm infection: a cross sectional study of a rural community in a subtropical, high rainfall area of Meghalaya, India. J. Parasitol. Dis. 22, 44–47. Matgor, R., Oku, Y., Gallardo, R., Yabzabal, L., 1996. High prevalence of Ancylostoma spp. infection in dogs associated with endemic focus of human cutaneous larvae migrans, in Tacuarembo, Uruguay. Parasite 3, 131–134. Nirmala, D., 1999. The effect of lead on immunity in mice to Ancylostoma caninum. M. Phil. Dissertation, Nagarjuna University, Nagarjuna Nagar, India. Sen, H.G., Joshi, U.N., Seth, D., 1965. Effect of cortisone upon Ancylostoma caninum infection in albino mice. Trans. R. Soc. Trop. Med. Hyg. 59, 684–689. Trehan, K., Maneesha, K., 1991. Effect of lead on nitrogenase and enzymes of nitrogen assimilation in a cyanobacterium Nostoc muscorum. Indian J. Exp. Biol. 29, 1116–1119. Vardhani, V.V., 1986. Serum levels of aspartate transaminase, alanine transaminase and worm burden in mice infected with Ancylostoma caninum larvae. Folia Parasitol. 33, 163–167.
Ecotoxicology and Environmental Safety 61 (2005) 134–136
Liver phosphatases in mice treated with lead during murine ancylostomiasis B.D.J. Satyalatha and V.V. Vardhani* Department of Zoology, Nagarjuna University, Nagarjuna Nagar (A.P.) 522 510, India Received 17 October 2002; received in revised form 12 December 2003; accepted 27 January 2004
Abstract Both infective larvae and pollutants disturb the homeostasis of female Swiss albino mice, causing ancylostomiasis and severe immunophysiological changes. In our experimental design, mice in group A were fed 0.01 mg of lead nitrate before infection with 500 larvae of Ancylostoma caninum, mice in group B were infected with larvae without previous lead treatment, and group C received lead nitrate treatment alone. Control animals in group D were neither treated with lead nitrate nor infected with larvae. While all three experimental groups showed disturbances in liver alkaline phosphatase (ALP) and acid phosphatase (ACP), group C receiving lead nitrate treatment alone and group A receiving lead nitrate with infection showed increased levels of ACP and decreased levels of ALP. r 2004 Elsevier Inc. All rights reserved. Keywords: Mice; Ancylostoma caninum; Liver; Phosphatases
1. Introduction Hookworm infection has long been known as one of the major cosmopolitan diseases of humanity. The zoonosis caused by canine hookworms is an interesting aspect to study because of the close association between humans and dogs (Lynden et al., 1998). Hookworm larvae penetrate the unbroken skin of humans and migrate through subcutaneous tissue. Since humans are not the natural hosts, the infective larvae are unable to complete their migratory cycle and produce skin lesions (Gilles, 1985). Human infection occurs in areas of recreational exposure to contaminated soil (Matgor et al., 1996). The effect of essential minerals and heavy metals (Co, Cu, Fe, Zn) and Ancylostoma caninum infection on the humoral and cellular components of immune response has been studied (Gowri and Vardhani, 1992; Ahmad et al., 1994; Boroskova et al., 1994). Mice exposed to oral doses of lead and challenged with a single dose of Hymenolepis nana eggs harbored significantly greater worm burden than did controls (Agrawal et al., 1989). The effect of heavy metals and/ or A. caninum infection on biochemical components of *Corresponding author. Fax: +91-863-2293378. E-mail address: [email protected] (V.V. Vardhani). 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.01.013
various experimental animals has been studied (Vardhani, 1986; Trehan and Maneesha, 1991; Nirmala, 1999; Lomte et al., 2000). In the case of humans, the contamination of air, food, and water (tropical diseases) led to ‘‘occupational diseases’’ caused by overexposure to a pollutant by virtue of an individual’s occupation. Some of the important contributors to environmental degradation are industrial effluents and automobile exhausts. Lead poisoning may occur in children and/or adults as a result of accidental or occupational ingestion. Lead is absorbed through the gastrointestinal tract or lungs; the absorbed lead is distributed into the liver, kidneys, and bone marrow, causing a direct toxic effect in these organs. Therefore, the present work was planned to study the effect of lead nitrate on liver phosphatases of female Swiss albino mice after oral treatment and/or infection.
2. Materials and methods Infective A. caninum larvae were cultured following the method of Sen et al. (1965). Mice in group A were treated orally with lead nitrate for 4 days (0.01 mg/ mouse) and infected with an oral dose of 500 larvae of
ARTICLE IN PRESS B.D.J. Satyalatha, V.V. Vardhani / Ecotoxicology and Environmental Safety 61 (2005) 134–136
135
Table 1 Liver enzymes, acid phosphatase, alkaline phosphatase values, and worm load in experimental mice at different times after infection. Day of necropsy
Experimental groups A
1
4
9
16
30
B
C
ACP (IU/L)
ALP (IU/L)
WL
ACP (IU/L)
ALP (IU/L)
WL
ACP (IU/L)
ALP (IU/L)
17.778.8 t ¼ 8:02 Po0:05 22.178.7 8.7 t ¼ 9:98 Po0:05 14.678.9 t ¼ 5:02 Po0:05 12.778.7 t ¼ 3:35 Po0:05 11. 378.8 t ¼ 1:70 P40:05
131.47159.7 t ¼ 2:02 P40:05 149.17159.6 t ¼ 0:68 P40:05 159.97159.7 t ¼ 0:10 P40:05 165.77159.8 t ¼ 3:62 P40:05 175.37159, 7 t ¼ 5:12 Po0:05
56 (11.2%)
15.278.8 t ¼ 5:02 Po0:05 14.378.7 t ¼ 4:90 Po0:05 11.878.9 t ¼ 1:89 P40:05 10.778.7 t ¼ 1:04 P40:05 9.678.8 t ¼ 0:98 P40:05
158.77159.7 t ¼ 0:02 P40:05 158.57159.7 t ¼ 0:96 P40:05 157.57159. 7 t ¼ 1:02 P40:05 157.47159.7 t ¼ 2:01 P40:05 156.97159.7 t ¼ 2:96 P40:05
58.0 (11.6%)
16.678.8 t ¼ 6:04 Po0:05 15.278.7 t ¼ 3:75 Po0:05 13.078.9 t ¼ 5:02 Po0:05 12.078.7 t ¼ 2:02 P40:05 11.078.8 t ¼ 1:96 P40:05
145.0 7159.7 t ¼ 2:08 Po0:05 1307159.6 t ¼ 2:20 Po0:05 1267159.7 t ¼ 2:14 Po0:05 124.67159.8 t ¼ 2:13 Po0:05 120.27159.7 t ¼ 2:24 Po0:05
50 (10.0%)
–
–
–
0.2 (0.4%)
–
–
–
Abbreviations: ACP, acid phosphatase; ALP, alkaline phosphatase; WL, worm load. Values are expressed as means derived from five observations.
A. caninum on day 5 (n ¼ 25), group B (n ¼ 25) with an equal amount of saline for 4 days and infected with an oral dose of 500 larvae on day 5, and group C (n ¼ 25) with lead nitrate alone (0.01 mg/mouse) for 4 days. Mice in group D were kept as untreated and uninfected controls. Five mice from each experimental group were sacrificed on day 1, 4, 9, 16, and 30 after infection (groups A and B) and lead nitrate treatment (group C) for the collection of larvae (from groups A and B) and for enzymatic assays from liver. The control mice were sacrificed for enzymatic assays on the same days as just described. The livers were excised and analyzed for phosphatases by the method described by Jaffee and Badansky (1943). Worm burden from liver was estimated from larval recoveries by Baermann’s technique.
3. Results and discussion The mean values of liver phosphatases from the experimental groups (A–C) and the control (group D) animals and worm load (groups A and B) for the 30-day experimental period with their statistical values are shown in Table 1. In group A mice (treated+infected), there was a considerable rise in acid phosphatase (ACP) from day 1 to 30, with a peak response on day 4 when compared with uninfected and untreated controls (group D); ACP levels then decreased gradually from that peak, reaching an almost normal value. The migration of larvae into the liver might have caused pathological reactions resulting in the overexpression of ACP. The increase was significant only when compared with controls (Po0:05). The amount of alkaline
phosphatase (ALP) in group A mice was below normal from day 1 to 16 but had significantly increased by day 30. This increase was found to be significant (Po0:05) in comparison with controls and with mice receiving the lead nitrate alone (group C), but not with mice infected with 500 larvae (group B). It is interesting to note that on day 30, the ACP value that had been high on day 1 decreased to a normal value, while the ALP value that had been below normal on day 1 increased significantly. In group B mice (untreated+infected), ACP values increased (Po0:05) from day 1 to 4 in comparison with controls, but not with mice receiving lead nitrate alone (group C); in these mice there was a gradual decrease of ACP from day 1 to 30. ALP activity was reduced slightly (P40:05) throughout the experimental period when compared with group D (there was a decrease in comparison with group C of below normal values). The activity of ALP remained almost stable during day 1 to 30. In group C mice receiving only lead, there was a twofold (Po0:05) rise in ACP from day 1 to 9 in comparison with controls; the ACP activity was not significant in comparison with groups A (treated+infected) and B (infected only). The increased amount of ACP (on day 1) decreased afterwards day by day, and the value on day 30 (11.0 units) was almost equal to that of group A (11.3 units). There was a below normal value (Po0:05) of ALP from day 1 to day 30 in comparison with controls and both experimental groups A and B. The decreased value of ALP on day 1 (in comparison with the control group) continued to decrease by B15% by day 30.
ARTICLE IN PRESS 136
B.D.J. Satyalatha, V.V. Vardhani / Ecotoxicology and Environmental Safety 61 (2005) 134–136
4. Statistical analysis ACP activities varied significantly in all the experimental groups when compared with controls. In group A, the values of ACP increased significantly from day 1 to 16 when compared with controls; from day 4, the ACP amount decreased gradually, reaching an almost nonsignificant level by day 30. Conversely, the values of ALP decreased to below normal levels from day 1 to 16 and increased significantly by day 30. In group B, ACP increased significantly on day 1 and 4, and there was a gradual decline from day 4 to day 30, reaching normal values by day 30. The retention of larval burden, increase of ACP on day 1 and 4, and the gradual decrease from day 4 to day 30 suggest an equivalent immune response in groups A and B. The decreased (group A) and stable (group B) nonsignificant values of ALP (except the significant value of group A on day 30) indicate that the hookworm infection may not alter the level of this enzyme. The nonsignificant values of ACP (except on days 1 and 4) and of ALP of group C suggest that a dose of 0.01 mg of lead nitrate may not bring about significant alterations in the levels of liver phosphatases. The larval burden in groups A and B suggests that adverse interactions took place between the host and lead and/or infective larvae, which in turn triggered a nonspecific reaction that disturbed the level of the liver enzymes. The results are comparable to Agrawal et al. (1989), who found increased worm burden in H. nana-infected mice exposed to low oral doses of lead. Increased larval burden is related to reduced immunity due to decreased globulin production (Griggs, 1964). The increased values of ALP (only from day 1 to 9 in group A) in experimental groups from 1 to 30 days of the experimental period indicate a correlation with the increased value of ACP. The increased value of ACP and decreased value of ALP suggest the role of lead and/ or infection. Lead may inhibit the enzyme d-aminolevulinic acid dehydrate, which is the most sensitive enzyme in the pathway, as suggested by Griggs (1964). Either a small dose of lead along with infection or lead alone resulted in decreased values of ALP and increased values of ACP in mice.
5. Conclusions The alteration of liver phosphatases (ACP and ALP) in mice treated with lead is comparable to that of Karthikeyan (2001), who also found cholestasis of the liver in rats treated with endosulfan. Our results indicate that the lead treatment alone and in combination with
hookworm infection induces toxic reactions in the liver, with subsequent disturbances in the metabolic–enzymatic homeostasis.
Acknowledgments We are thankful to Lutheran World Federation, Geneva for providing funds to one of us (B.D.J. Satyalatha) to carry out this work.
References Agrawal, R., Chowdary, S., Johri, G.N., 1989. Effect of lead on the retention of parasitic burden and certain hematological changes in the Swiss albino mice, Mus musculus albinos. Proc. Natl. Acad. Sci. USA 59, 33–37. Ahmad, S., Singh, V., Rao, G.S., 1994. Antioxidant potential in serum and liver of albino rats exposed to benzene. Indian J. Exp. Biol. 32, 203–206. Boroskova, Z., Benkova, M., Soltys, J., Krupicer, I., 1994. Effect of heavy metal emission on the immune response of sheep with experimental fasciolosis. Parasite Immunol. 16, 389–391. Gilles, H.M., 1985. Selective primary health care strategies for control of disease in the developing world. XVII. Hookworm infection and anaemia. Rev. Infect. Dis. 7, 111–118. Gowri, P., Vardhani, V.V., 1992. Eosinophilia in murine ancylostomiasis. J. Hyg. Epidemiol. Microbiol. Immunol. 36, 175–180. Griggs, R.C., 1964. Lead poisoning: hematological aspects. In: Progress in Hematology, Grune and Stratton, New York, p. 117. Jaffee, H.C., Badansky, O., 1943. Diagnostic significance of serum alkaline and acid phosphatase values in relation to bone disease. Bull. NY Acad. Med. 19, 831–848. Karthikeyan, S., 2001. Effect of chronic administration of endosulfan on transaminases, alkaline phosphatase value in relation to bone disease. J. Ecotoxicol. Environ. Monit. 1 (3), 169–172. Lomte, V.S., Rajendra, T.C., Sultana, M., 2000. Changes in protein content of fresh water Bivalve, Parreysia cylindrica on exposure to nickel chloride and lead chloride. Indian J. Ecoenviron. Ecoplanning 3, 63–65. Lynden, L.M., Tandorc, V., Yadav, A.K., 1998. Hookworm infection: a cross sectional study of a rural community in a subtropical, high rainfall area of Meghalaya, India. J. Parasitol. Dis. 22, 44–47. Matgor, R., Oku, Y., Gallardo, R., Yabzabal, L., 1996. High prevalence of Ancylostoma spp. infection in dogs associated with endemic focus of human cutaneous larvae migrans, in Tacuarembo, Uruguay. Parasite 3, 131–134. Nirmala, D., 1999. The effect of lead on immunity in mice to Ancylostoma caninum. M. Phil. Dissertation, Nagarjuna University, Nagarjuna Nagar, India. Sen, H.G., Joshi, U.N., Seth, D., 1965. Effect of cortisone upon Ancylostoma caninum infection in albino mice. Trans. R. Soc. Trop. Med. Hyg. 59, 684–689. Trehan, K., Maneesha, K., 1991. Effect of lead on nitrogenase and enzymes of nitrogen assimilation in a cyanobacterium Nostoc muscorum. Indian J. Exp. Biol. 29, 1116–1119. Vardhani, V.V., 1986. Serum levels of aspartate transaminase, alanine transaminase and worm burden in mice infected with Ancylostoma caninum larvae. Folia Parasitol. 33, 163–167.