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Effect of Lead and Ancylostomiasis on Phosphatases of Heart in Swiss Albino Mice
Ecotoxicology and Environmental Safety 53, 281}284 (2002) Environmental Research, Section B doi:10.1006/eesa.2002.2217
Effect of Lead and Ancylostomiasis on Phosphatases of Heart in Swiss Albino Mice B. D. J. Satya Latha and V. V. Vardhani Department of Zoology, Nagarjuna University, Nagarjuna Nagar (A.P.) 522 510, India Received November 1, 2001
Administration of 0.01 and 0.1 mg of lead nitrate for 4 and 7 days and infection of Ancylostoma caninum larvae orally altered the activation of alkaline phosphatase and acid phosphatase in the hearts of mice when compared to infected animals and controls. Alkaline phosphatase activity increased signi5cantly in all drug-treated#infected mice. The level of acid phosphatase decreased signi5cantly in mice exposed to chronic doses of lead. The altered levels of alkaline phosphatase and acid phosphatase suggest that administration of lead could cause toxicity in the heart, disturbing the cellular metabolism; infection alone could not cause any signi5cant changes in enzymes of heart. 2002 Elsevier Science (USA) Key Words: lead; Ancylostoma caninum; phosphatases; mice. INTRODUCTION
In recent experimental studies, the e!ects of heavy metals on natural immunity and/or physiological changes have received much attention with regard to enzymatic changes. Similar studies have been reported with infection of Hymenolepis nana and lead treatment in mice (Agrawal et al., 1989). A single intracardiac injection of lead acetate caused abnormal DNA and RNA synthesis in mouse kidney (Fairchild, 1978). A high degree of abnormalties was found in tissue nucleic acids of rats treated with lead (Chakraborty et al., 1988). Tissue response to Ancylostoma caninum larvae and toxic e!ect of lead on immunophysiology in mice has been studied by several workers (Griggs, 1964; Brown, 1980; Vardhani, 1986; Nirmala and Vardhani, 2001). Alteration of phosphatase levels was observed in liver diseases, tumors, and other abnormalities of cardiovascular system. The present investigation was undertaken to estimate the level of phosphatses in heart at various doses of lead and infection and to assess their importance in the mouse system. MATERIALS AND METHODS
Infective A. caninum larvae were cultured following the method of Sen et al. (1965). Female Swiss albino mice (6 to
8 weeks old, 23}26 g) were used in this work. Thirty mice were treated orally with 0.01 mg of lead nitrate/mouse/4 days (group A), 30 with 0.01 mg/mouse/7 days (group B), 30 with 0.1 mg/mouse/4 days (group C), and 30 with 0.1 mg/mouse/7 days (group D). These animals were inoculated with 500 larvae of A. caninum on Days 5 (groups A and C) and 8 (groups B and D) after lead treatmet. Thirty mice were untreated but infected with 500 larvae (group E). One hundred "fty mice were kept as controls for comparison (group F). Five mice from each experimental and control group were sacri"ced on Days 1,4,9,16, and 30 of infection (5 days after infection in groups A and C; 8 days after in groups B and D) for the collection of heart and larvae. The hearts were quickly separated and the activities of alkaline phosphatase (ALP) and acid phosphatase (ACP) were assayed by the methods of Ja!ee and Badansky (1943). Values were expressed as units per gram wet tissue. Results were subjected to Student's t test (Lewis, 1966). RESULTS AND DISCUSSION
Alkaline phosphatase and acid phosphatase are used as marker enzymes. Altered levels can indicate cardiac toxicity. Results are revealed in Figs. 1 and 2 and Table 1. Figure 1 reveals a signi"cant rise in ACP in mice that received 0.1 mg of lead for 4 (group C) and 7 (group D) consecutive days and infected with 500 larvae of A. caninum. In comparison with controls, the levels of ACP and ALP were found to be nonsigni"cant (P(0.05) in the in mice that received infection alone (group E). These results suggest that the toxicant might have caused adverse changes in the levels of both ALP and ACP. There is also indication that A. caninum infection alone may not alter the level of heart phosphatases. The gradual decrease of ACP (from days 4 to 30) and ALP (from Days 1 to 30) in the heart (the blood pumping organ) of all experimental groups might be due to anemia caused by lead toxication and/or ancylostomiasis (Fig. 2) Qayyam and Shammi (1983) and Goel et al. (1984) have reported that these toxicants can cause anemia.
281 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
282
SATYA LATHA AND VARDHANI
FIG. 1. Comparison of alkaline phosphatase (ALP) activity in the heart of various groups of treated (mice inoculated with di!erent concentrations of lead #500 larvae of Ancylostoma caninum), infected (mice untreated with lead but inoculated with 500 larvae of A. caninum), and control (untreated#uninfected). *Group A, 0.01 mg/mouse/4 days#infected; Group B, 0.01 mg/mouse/7 days#infected; Group C, 0.1 mg/mouse/4 days#infected; Group D, 0.1 mg/mouse/7 days#infected; Group E, untreated but infected; Group F, untreated and uninfected (control).
A. caninum infection in mice causes varying degrees of anemia, hypoproteinemia (hypoalbuminemia), bloody diarrhea, wasting, and death (Vardhani, 1976). The presence of A. caninum hookworm L larvae in the mouse intestine causes damage that leads to bleeding and excessive blood loss leads to anemia (Vardhani, 1976). In parasitism or under stress factors, the body of the host demands high energy; a number of hematologic and physiologic changes occur in the body of the host. Thus, in the present investigations the signi"cant rise of alkaline phosphatase in lead-treated animals re#ects heart cell injury
and the nonsigni"cant activity of this enzyme (ACP) in infected animals and controls further substantiates this observation. Karthikeyan (2001) found elevated levels of alkaline phosphatase in serum of rats treated with endosulfan. The mean values of enzymes with their t values for the 30-day experimental period are found in Table 2. ALP levels were statistically signi"cant in lead-treated#infected groups A,B,C, and D compared with those of infected animals (E) and controls (F). ACP levels in groups A and E was nonsigni"cant compared with controls. There was no
FIG. 2. Comparison of acid phosphatase (ACP) activity in the heart of various groups of treated (mice inoculated with di!erent concentrations of lead #500 larvae of Ancylostoma caninum), infected (mice untreated with lead but inoculated with 500 larvae of A. caninum), and control (untreated#uninfected). *Group A, 0.01 mg/mouse/4 days#infected; Group B, 0.01 mg/mouse/7 days#infected; Group C, 0.1 mg/mouse/ 4 days# infected; Group D, 0.1 mg/mouse/7 days#infected; Group E, untreated but infected; Group F, untreated and uninfected (control).
283
EFFECTS OF Pb AND ANCYLOSTOMIASIS ON MOUSE HEART
TABLE 1 Alkaline Phosphatase (ALP) and Acid Phosphatase (ACP) in Heart of Control (Group F), Treated (Groups A,B,C, and D), and Infected (Group E) Mice at Di4erent Periods of Infection
Days of Necro psy 1 4 9 16 30
Group A (0.01 mg/4 days and infected with 500 larvae)
Group B (0.01 mg/7 days and infected with 500 larvae)
Group C (0.1 mg/4 days and infected with 500 larvae)
Group D (0.1 mg/7 days and infected with 500 larvae)
Group E (Untreated but infected with 500 larvae)
Group F (Untreated and uninfected)
ALP
ACP
ALP
ACP
ALP
ACP
ALP
ACP
ALP
ACP
ALP
ACP
20.0 18.2 16.3 15.9 14.1
3.2 5.7 4.7 3.5 3.2
20.6 18.8 16.6 15.8 14.1
3.8 5.8 4.6 4.4 3.2
29.0 27.2 25.3 16.8 14.8
6.1 8.6 7.8 6.5 5.6
64.1 50.7 37.1 22.1 21.3
4.4 8.8 7.5 6.8 5.8
10.7 10.6 10.7 10.6 10.4
2.8 2.7 2.7 2.6 2.5
10.6 10.7 10.8 10.7 10.5
2.9 2.7 2.7 2.8 2.9
Note. Values are expressed as mean derived from "ve observations.
TABLE 2 Mean Values of Phosphatases with t Values Obtained for Hearts of Experimental and Control Mice Experimental group A Enzymes: 0.01 mg/4 days# infected Alkaline phosphatase: 16.9
B 0.01 mg/ 7 days# infected 17.1
C 0.1 mg/ 4 days
D 0.1 mg/ 7 days
E Untreated #infected
F Control
22.6
39.0
10.5
10.6
A F B F C F D F " " " " " " " " **** **** **** **** 6.21* 5.70* 4.15* 3.42* (P'0.05) (P'0.05) (P'0.05) (P'0.05) A B A C A D A E " " " " " " " " **** **** **** **** 0.18? 3.20* 12.41* 6.22* (P(0.05) (P'0.05) (P'0.05) (P'0.05) B C B D B E " " " " " " **** **** **** 1.13? 2.61* 3.68* (P(0.05) (P'0.05) (P'0.05) C D C E D E " " " " " " **** **** **** 1.87? 6.73* 15.94* (P(0.05) (P'0.05) (P'0.05) Acid phosphatase: 4.06
4.36
A F B F " " " " **** **** 2.18? 3.52* (P'0.05) (P'0.05) A B A C " " " " **** **** 0.45? 3.86* (P(0.05) (P'0.05) B C " " **** 1.27? (P(0.05) C D " " **** 0.27? (P(0.05) Note. P value at 5% level of signi"cance is 2.306. *Statistically signi"cant values. ?Statistically signi"cant values.
6.90 C F " " **** 7.40* (P'0.05) A D " " **** 2.91* (P'0.05) B D " " **** 7.62* (P'0.05) C E " " **** 7.62* (P'0.05)
6.60 D F " " **** 5.12* (P'0.05)
2.50
E F " " **** 1.54? (P(0.05) A E " " **** 2.89* (P'0.05) B E " " **** 5.35* (P'0.05) D E " " **** 5.35* (P'0.05)
E F " " **** 0.41? (P(0.05)
2.86
284
SATYA LATHA AND VARDHANI
signi"cant di!erence in ALP and ACP level among groups A and B, B and C, and C and D. The signi"cant increase in ALP and the steep decline in ACP indicates altered cellular metabolism in tested mice. Transaminase changes due to ancylostomiasis have been reported to be an index of biochemical, morphologic, or functional alterations of tissues (Vardhani, 1986). In the present study, the signi"cant alteration in alkaline phosphatase with 1.0 mg/mouse/7 day treatment suggests that chronic administration of lead induces cardiotoxicity in mice. Low levels of phosphatases in heart with 0.01 mg/4 days and 0.01 mg/7 days suggests that administration of small doses of lead does not induce signi"cant toxicity. CONCLUSION
The elevation of alkaline phosphatase resulting from chronic doses and of lead and the simultaneous decline from low doses is similar to that found by Kartihikeyan (2001), who reported endosulfan-induced cholestiasis of the liver in rats. The results of this study suggest that lead treatment induces changes in the metabolism of phosphatases in the heart of mice. ACKNOWLEDGMENT This work was supported by a grant from the LWF, Geneva, to one of the authors (B.D.J.S).
REFERENCES Agrawal, R., Choudhary, S., and Johri, G. N. (1989). E!ect of lead on the retention of parasitic burden and haematological changes in the
Swiss albino mice, Mus musculus albinus. Proc. Natl. Acad. Sci. India 59, 33}77. Brown, V. K. (1980). Acute ¹oxicity in ¹heory and Practice. Wiley, Norwich. Chakraborty, I., Talukder, G., and Sharma, A. (1988). E!ects of inorganic lead on cell division and nucleic acids in rats. Proc. Natl. Acad. Sci. India 58, 11. Fairchild, E. J. (1978). Agricultural Chemicals and Pesticides. A Handbook of the ¹oxic E+ects. Castle House Publications Ltd., New Delhi. Goel, A. K., Kalpana, S., and Agrawal, V. P. (1984). Alachlor toxicity to a fresh water teleost, Clarius batrachus. Curr. Sci. 58, 1050}1052. Griggs, R. C. (1964). Lead poisoning: Haematologic aspects. In Progress in Haematology, p. 117. Grune & Stration, New York. Ja!ee, H. C., and Badansky, O. (1943). Diagnostic signi"cance of serum alkaline and acid phosphatase value in relation to bone disease. Bull. =. >. Acad. Med. 19, 831}848. Karthikeyan, S. (2001). E!ect of chronic administration of endosulfan on transaminases, alkaline phosphatase and protein in serum and liver of albino rats. J. Ecotoxicol. Environ. Monit. 1(3), 169}172. Lewis, E. A. (1966). Biostatistics. A$liated East West Press Pvt. Ltd., New Delhi, India. Nirmala, D., and Vardhani, V. V. (2001). The e!ect of lead on natural immunity during ancylostomiasis (in press). Qayyum, M. A., and Shammi, G. J. (1983). Haematological values of a teleost "sh, Catla catla, exposed to aldrin. J. Sci. Res. 5, 129}132. Sen, H. G., Joshi, U. N., and Seth, D. (1965). E!ect of cortisone upon Ancylostoma caninum infection in albino mice. ¹rans. R. Soc. ¹rop. Med. Hyg. 59, 684}689. Vardhani, V. V. (1976). Weight loss, leucocytic counts and immunologically injured peritoneal cell counts in mice infected with Ancylostoma caninum larvae.
Effect of Lead and Ancylostomiasis on Phosphatases of Heart in Swiss Albino Mice B. D. J. Satya Latha and V. V. Vardhani Department of Zoology, Nagarjuna University, Nagarjuna Nagar (A.P.) 522 510, India Received November 1, 2001
Administration of 0.01 and 0.1 mg of lead nitrate for 4 and 7 days and infection of Ancylostoma caninum larvae orally altered the activation of alkaline phosphatase and acid phosphatase in the hearts of mice when compared to infected animals and controls. Alkaline phosphatase activity increased signi5cantly in all drug-treated#infected mice. The level of acid phosphatase decreased signi5cantly in mice exposed to chronic doses of lead. The altered levels of alkaline phosphatase and acid phosphatase suggest that administration of lead could cause toxicity in the heart, disturbing the cellular metabolism; infection alone could not cause any signi5cant changes in enzymes of heart. 2002 Elsevier Science (USA) Key Words: lead; Ancylostoma caninum; phosphatases; mice. INTRODUCTION
In recent experimental studies, the e!ects of heavy metals on natural immunity and/or physiological changes have received much attention with regard to enzymatic changes. Similar studies have been reported with infection of Hymenolepis nana and lead treatment in mice (Agrawal et al., 1989). A single intracardiac injection of lead acetate caused abnormal DNA and RNA synthesis in mouse kidney (Fairchild, 1978). A high degree of abnormalties was found in tissue nucleic acids of rats treated with lead (Chakraborty et al., 1988). Tissue response to Ancylostoma caninum larvae and toxic e!ect of lead on immunophysiology in mice has been studied by several workers (Griggs, 1964; Brown, 1980; Vardhani, 1986; Nirmala and Vardhani, 2001). Alteration of phosphatase levels was observed in liver diseases, tumors, and other abnormalities of cardiovascular system. The present investigation was undertaken to estimate the level of phosphatses in heart at various doses of lead and infection and to assess their importance in the mouse system. MATERIALS AND METHODS
Infective A. caninum larvae were cultured following the method of Sen et al. (1965). Female Swiss albino mice (6 to
8 weeks old, 23}26 g) were used in this work. Thirty mice were treated orally with 0.01 mg of lead nitrate/mouse/4 days (group A), 30 with 0.01 mg/mouse/7 days (group B), 30 with 0.1 mg/mouse/4 days (group C), and 30 with 0.1 mg/mouse/7 days (group D). These animals were inoculated with 500 larvae of A. caninum on Days 5 (groups A and C) and 8 (groups B and D) after lead treatmet. Thirty mice were untreated but infected with 500 larvae (group E). One hundred "fty mice were kept as controls for comparison (group F). Five mice from each experimental and control group were sacri"ced on Days 1,4,9,16, and 30 of infection (5 days after infection in groups A and C; 8 days after in groups B and D) for the collection of heart and larvae. The hearts were quickly separated and the activities of alkaline phosphatase (ALP) and acid phosphatase (ACP) were assayed by the methods of Ja!ee and Badansky (1943). Values were expressed as units per gram wet tissue. Results were subjected to Student's t test (Lewis, 1966). RESULTS AND DISCUSSION
Alkaline phosphatase and acid phosphatase are used as marker enzymes. Altered levels can indicate cardiac toxicity. Results are revealed in Figs. 1 and 2 and Table 1. Figure 1 reveals a signi"cant rise in ACP in mice that received 0.1 mg of lead for 4 (group C) and 7 (group D) consecutive days and infected with 500 larvae of A. caninum. In comparison with controls, the levels of ACP and ALP were found to be nonsigni"cant (P(0.05) in the in mice that received infection alone (group E). These results suggest that the toxicant might have caused adverse changes in the levels of both ALP and ACP. There is also indication that A. caninum infection alone may not alter the level of heart phosphatases. The gradual decrease of ACP (from days 4 to 30) and ALP (from Days 1 to 30) in the heart (the blood pumping organ) of all experimental groups might be due to anemia caused by lead toxication and/or ancylostomiasis (Fig. 2) Qayyam and Shammi (1983) and Goel et al. (1984) have reported that these toxicants can cause anemia.
281 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
282
SATYA LATHA AND VARDHANI
FIG. 1. Comparison of alkaline phosphatase (ALP) activity in the heart of various groups of treated (mice inoculated with di!erent concentrations of lead #500 larvae of Ancylostoma caninum), infected (mice untreated with lead but inoculated with 500 larvae of A. caninum), and control (untreated#uninfected). *Group A, 0.01 mg/mouse/4 days#infected; Group B, 0.01 mg/mouse/7 days#infected; Group C, 0.1 mg/mouse/4 days#infected; Group D, 0.1 mg/mouse/7 days#infected; Group E, untreated but infected; Group F, untreated and uninfected (control).
A. caninum infection in mice causes varying degrees of anemia, hypoproteinemia (hypoalbuminemia), bloody diarrhea, wasting, and death (Vardhani, 1976). The presence of A. caninum hookworm L larvae in the mouse intestine causes damage that leads to bleeding and excessive blood loss leads to anemia (Vardhani, 1976). In parasitism or under stress factors, the body of the host demands high energy; a number of hematologic and physiologic changes occur in the body of the host. Thus, in the present investigations the signi"cant rise of alkaline phosphatase in lead-treated animals re#ects heart cell injury
and the nonsigni"cant activity of this enzyme (ACP) in infected animals and controls further substantiates this observation. Karthikeyan (2001) found elevated levels of alkaline phosphatase in serum of rats treated with endosulfan. The mean values of enzymes with their t values for the 30-day experimental period are found in Table 2. ALP levels were statistically signi"cant in lead-treated#infected groups A,B,C, and D compared with those of infected animals (E) and controls (F). ACP levels in groups A and E was nonsigni"cant compared with controls. There was no
FIG. 2. Comparison of acid phosphatase (ACP) activity in the heart of various groups of treated (mice inoculated with di!erent concentrations of lead #500 larvae of Ancylostoma caninum), infected (mice untreated with lead but inoculated with 500 larvae of A. caninum), and control (untreated#uninfected). *Group A, 0.01 mg/mouse/4 days#infected; Group B, 0.01 mg/mouse/7 days#infected; Group C, 0.1 mg/mouse/ 4 days# infected; Group D, 0.1 mg/mouse/7 days#infected; Group E, untreated but infected; Group F, untreated and uninfected (control).
283
EFFECTS OF Pb AND ANCYLOSTOMIASIS ON MOUSE HEART
TABLE 1 Alkaline Phosphatase (ALP) and Acid Phosphatase (ACP) in Heart of Control (Group F), Treated (Groups A,B,C, and D), and Infected (Group E) Mice at Di4erent Periods of Infection
Days of Necro psy 1 4 9 16 30
Group A (0.01 mg/4 days and infected with 500 larvae)
Group B (0.01 mg/7 days and infected with 500 larvae)
Group C (0.1 mg/4 days and infected with 500 larvae)
Group D (0.1 mg/7 days and infected with 500 larvae)
Group E (Untreated but infected with 500 larvae)
Group F (Untreated and uninfected)
ALP
ACP
ALP
ACP
ALP
ACP
ALP
ACP
ALP
ACP
ALP
ACP
20.0 18.2 16.3 15.9 14.1
3.2 5.7 4.7 3.5 3.2
20.6 18.8 16.6 15.8 14.1
3.8 5.8 4.6 4.4 3.2
29.0 27.2 25.3 16.8 14.8
6.1 8.6 7.8 6.5 5.6
64.1 50.7 37.1 22.1 21.3
4.4 8.8 7.5 6.8 5.8
10.7 10.6 10.7 10.6 10.4
2.8 2.7 2.7 2.6 2.5
10.6 10.7 10.8 10.7 10.5
2.9 2.7 2.7 2.8 2.9
Note. Values are expressed as mean derived from "ve observations.
TABLE 2 Mean Values of Phosphatases with t Values Obtained for Hearts of Experimental and Control Mice Experimental group A Enzymes: 0.01 mg/4 days# infected Alkaline phosphatase: 16.9
B 0.01 mg/ 7 days# infected 17.1
C 0.1 mg/ 4 days
D 0.1 mg/ 7 days
E Untreated #infected
F Control
22.6
39.0
10.5
10.6
A F B F C F D F " " " " " " " " **** **** **** **** 6.21* 5.70* 4.15* 3.42* (P'0.05) (P'0.05) (P'0.05) (P'0.05) A B A C A D A E " " " " " " " " **** **** **** **** 0.18? 3.20* 12.41* 6.22* (P(0.05) (P'0.05) (P'0.05) (P'0.05) B C B D B E " " " " " " **** **** **** 1.13? 2.61* 3.68* (P(0.05) (P'0.05) (P'0.05) C D C E D E " " " " " " **** **** **** 1.87? 6.73* 15.94* (P(0.05) (P'0.05) (P'0.05) Acid phosphatase: 4.06
4.36
A F B F " " " " **** **** 2.18? 3.52* (P'0.05) (P'0.05) A B A C " " " " **** **** 0.45? 3.86* (P(0.05) (P'0.05) B C " " **** 1.27? (P(0.05) C D " " **** 0.27? (P(0.05) Note. P value at 5% level of signi"cance is 2.306. *Statistically signi"cant values. ?Statistically signi"cant values.
6.90 C F " " **** 7.40* (P'0.05) A D " " **** 2.91* (P'0.05) B D " " **** 7.62* (P'0.05) C E " " **** 7.62* (P'0.05)
6.60 D F " " **** 5.12* (P'0.05)
2.50
E F " " **** 1.54? (P(0.05) A E " " **** 2.89* (P'0.05) B E " " **** 5.35* (P'0.05) D E " " **** 5.35* (P'0.05)
E F " " **** 0.41? (P(0.05)
2.86
284
SATYA LATHA AND VARDHANI
signi"cant di!erence in ALP and ACP level among groups A and B, B and C, and C and D. The signi"cant increase in ALP and the steep decline in ACP indicates altered cellular metabolism in tested mice. Transaminase changes due to ancylostomiasis have been reported to be an index of biochemical, morphologic, or functional alterations of tissues (Vardhani, 1986). In the present study, the signi"cant alteration in alkaline phosphatase with 1.0 mg/mouse/7 day treatment suggests that chronic administration of lead induces cardiotoxicity in mice. Low levels of phosphatases in heart with 0.01 mg/4 days and 0.01 mg/7 days suggests that administration of small doses of lead does not induce signi"cant toxicity. CONCLUSION
The elevation of alkaline phosphatase resulting from chronic doses and of lead and the simultaneous decline from low doses is similar to that found by Kartihikeyan (2001), who reported endosulfan-induced cholestiasis of the liver in rats. The results of this study suggest that lead treatment induces changes in the metabolism of phosphatases in the heart of mice. ACKNOWLEDGMENT This work was supported by a grant from the LWF, Geneva, to one of the authors (B.D.J.S).
REFERENCES Agrawal, R., Choudhary, S., and Johri, G. N. (1989). E!ect of lead on the retention of parasitic burden and haematological changes in the
Swiss albino mice, Mus musculus albinus. Proc. Natl. Acad. Sci. India 59, 33}77. Brown, V. K. (1980). Acute ¹oxicity in ¹heory and Practice. Wiley, Norwich. Chakraborty, I., Talukder, G., and Sharma, A. (1988). E!ects of inorganic lead on cell division and nucleic acids in rats. Proc. Natl. Acad. Sci. India 58, 11. Fairchild, E. J. (1978). Agricultural Chemicals and Pesticides. A Handbook of the ¹oxic E+ects. Castle House Publications Ltd., New Delhi. Goel, A. K., Kalpana, S., and Agrawal, V. P. (1984). Alachlor toxicity to a fresh water teleost, Clarius batrachus. Curr. Sci. 58, 1050}1052. Griggs, R. C. (1964). Lead poisoning: Haematologic aspects. In Progress in Haematology, p. 117. Grune & Stration, New York. Ja!ee, H. C., and Badansky, O. (1943). Diagnostic signi"cance of serum alkaline and acid phosphatase value in relation to bone disease. Bull. =. >. Acad. Med. 19, 831}848. Karthikeyan, S. (2001). E!ect of chronic administration of endosulfan on transaminases, alkaline phosphatase and protein in serum and liver of albino rats. J. Ecotoxicol. Environ. Monit. 1(3), 169}172. Lewis, E. A. (1966). Biostatistics. A$liated East West Press Pvt. Ltd., New Delhi, India. Nirmala, D., and Vardhani, V. V. (2001). The e!ect of lead on natural immunity during ancylostomiasis (in press). Qayyum, M. A., and Shammi, G. J. (1983). Haematological values of a teleost "sh, Catla catla, exposed to aldrin. J. Sci. Res. 5, 129}132. Sen, H. G., Joshi, U. N., and Seth, D. (1965). E!ect of cortisone upon Ancylostoma caninum infection in albino mice. ¹rans. R. Soc. ¹rop. Med. Hyg. 59, 684}689. Vardhani, V. V. (1976). Weight loss, leucocytic counts and immunologically injured peritoneal cell counts in mice infected with Ancylostoma caninum larvae.