Suppression of the hepatic microsomal cytochrome P-450 dependent mixed function oxidase. Activities in golden hamster during Leishmania donovani infection

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SUPPRESSION OF THE HEPATIC MICROSOMAL CYTOCHROME P-450 DEPENDENT MIXED FUNCTION OXIDASE. ACTIVITIES IN GOLDEN HAMSTER DURING Leishmania donovani INFECTION A. K . SINGH*, B. L . TEKWANI*, P. Y. GURUt, A . K. RASTOGI*, and V. C. PANDEY* Divisions of *Biochemistry and tParasitology, Central Drug Research Institute, P.O. Box 173, Lucknow 226 001 (UP), India Received in final form 15 February 1989

SUMMARY Experimental infection of golden hamsters with Leishmania donovani caused significant alterations in the hepatic microsomal mixed function oxidase system . Gross examination of liver indicated hepatomegaly . Microsomal protein contents were only marginally elevated . Cytochrome P-450 as well as haem contents were significantly decreased and it directly correlated with the degree of infection . Cytochrome b5 exhibited elevation at lower degrees of infection which came down to control levels at the peak infection . Concomitant suppression was also noticed in cytochrome P-450 dependent monooxygenase activities, viz . aniline hydroxylase, benzo[a]pyrene hydroxylase and aminopyrine N-demethylase . No significant change was observed in NADH-cytochrome b 5 reductase and NADPHcytochrome c reductase . The results indicate suppression of hepatic microsomal MFO activities during visceral leishmaniasis . KEY woiDs: Leishmania donovani,

cytochrome P-450, liver, microsomes .

INTRODUCTION The hepatic cytochrome P-450 dependent mixed function oxidase (MFO) system plays an important role in the biotransformation of xenobiotics, viz . drugs, carcinogens and environmental pollutants [1], as well as several endogenous compounds, viz . steroids, fatty acids, prostaglandins, cholesterol and bile acids [2] . The status of this system is modulated by species, sex, hormonal, nutritional and environmental factors [3, 4] . The depression in MFO activities has been reported during hepatitis [5], active viral [6] and bacterial infections [7] as well as infection by tropical disease pathogens [8] . However, no reports are yet available regarding status of this system in the host during visceral leishmaniasis or kala-azar, a fatal disease caused by a protozoan parasite, Leishmania donovani. The 1043-6618/89/050507-06/$03 .00/0

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histopathological lesions in fatal infections of L. donovani in man are characterized by macrophage infiltration in most host organs . Liver is one of the primary sites of infection [9] . The organ is greatly enlarged and congested . The Kupffer cells are increased in size and number, their cytoplasma are packed with the amastigote form of L. donovani . The sinusoidal capillaries are dilated and engorged with blood . However, the hepatocytes remain free from parasitization but these cells also undergo thinning and atrophy due to pressure of dilated sinusoidal capillaries . A certain amount of fatty change also occurs in the liver . Reticuloendothelial proliferation in hepatic tissue is a common feature of visceral leishmaniasis [10] . Under laboratory conditions, L . donovani infects many animal species, responding in a manner similar to that seen in fatal human infections [111 . Infection of golden hamsters (Mesocricetus auratus) with L . donovani is a useful model to study the pathogenesis of the disease [12] . The status of the hepatic MFO system in hamsters infected with L. donovani is reported, describing the possible consequences of the derangement . METHODS Parasite and the experimental host

The HOM/IN/80 Dd-8 strain of L. donovani originally obtained from Prof. P.C .C . Garnham, Imperial College, London was maintained in golden hamsters by serial intracardial inoculations . Male golden hamsters, weighing about 40-50 g, obtained from the Division of Laboratory Animals, CDRI, Lucknow, were used as experimental hosts in the present studies . Each hamster was injected intracardially with about 10 million amastigotes of L . donovani, prepared from the spleen of previously infected hamsters . The parasite migrates from heart to different regions rich in fibrous elements like liver, spleen and bone marrow, through the blood circulation . Liver and spleen of infected hamsters were excised out and smears were made on the microscopic slides, fixed with methanol and stained with Giemsa stain (10%) for 30 min . The degree of infection was monitored under light microscope and expressed in terms of number of parasites (amastigotes) present per 100 cell nuclei . Preparation of hepatic microsomes

Liver obtained from control as well as L . donovani infected hamsters were washed with chilled normal saline freed from blood clots, blotted carefully before weighing and homogenized in chilled 150 mM KCl (20% w/v) buffered with PO, buffer (50 mm, pH 7 . 6) . The homogenates were centrifuged at 11 000 g (20 min) . An aliquot of the supernatant (post mitochondrial fraction, PMF) was retained for the assay of cytochrome P-450 dependent monooxygenases . Remaining fraction was further centrifuged at 105 000 g (60 min) in a Beckman ultracentrifuge model L5-65B . The microsomal pellet was washed once with buffered KCl and finally suspended in phosphate buffer (50 mm, pH 7 . 6) containing EDTA (1 mm) . Both the fractions, i .e . PMF and isolated microsomes, were immediately used for biochemical assays .



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Biochemical assays

The enzymes aminopyrine N-demethylase [13], aniline hydroxylase [14] and benzo[a]pyrene hydroxylase [15] were assayed in PMF while cytochrome P-450, cytochrome b5 [14] and haem contents as well as NADPH- cytochrome c reductase [17], and NADH-cytochrome b5 reductase [18] activities were measured in isolated microsomes . Protein was measured according to Lowry et al. [19] using BSA as standard . Statistical analysis

The values of infected and control groups were compared by Student's t-test and P values less than 0 . 05 (5%) were considered as significant .

RESULTS Initially some gross parameters, microsomal cytochrome P-450 contents as well as some associated components, viz . cytochrome b5 and haem, were evaluated in the hamsters exhibiting various degrees of L. donovani infection in hepatic tissues . The degree of the infection was expressed in terms of number of amastigotes present per 100 cell nuclei. A sequential increase in total liver weight was noticed . Microsomal protein contents were only marginally elevated (Table I) . The hamsters Table I Gross alteration in liver of golden hamsters during L. donovani infection Parameter

Total liver weight (g) Microsomal protein (mg g - ' fresh tissue)

Control

Degree of infection 4-10%

30-70%

> 100%

3 . 27 ± 0 . 42

3 .73 ± 0 . 49

4. 92 ± 0 . 52*

5 . 20 ± 0 . 42*

19 . 80 ± 2 . 61

21 . 71 ±3-10

23 . 14 ±3-91

22 . 91 ±2-91

Values are mean ± SD of three separate observations from separate animals. *Statistically significant change over controls . registering mild infection (4-10%) did not exhibit change in cytochrome P450 and haem . Cytochrome b5 contents showed marginal increase at lower degrees of the infection which was statistically insignificant . At more than 30% degree of the infection significant decrease in cytochrome b5 content was observed. However, the change in cytochrome b5 was again statistically insignificant at more than 100% degree of the infection (Fig . 1) . At higher degrees of infection, cytochrome P-450 as well as haem contents decreased significantly . However, the decrease was



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Cytochrome

Cytochrome

b5

Haem

P-450

o o0 U

0 .623± 0-073'

1 .267± 0 .147

EJ Control 0 4-10% 030-70% 0 > 100%

2 .960± 0 .413

o N oO G) U a, Û_

Degree of infection

Fig. 1 . Changes in hepatic microsomal cytochrome b5 , cytochrome P-450 and haem contents of golden hamsters during experimental infection with Leishmania donovani. Values given over blank bars are control levels and are mean ± SD of six observations from separate animals, expressed as nmol (mg protein) -1 . Per cent values in experimental samples were calculated taking control values as 100 . *Statistically significant change as compared to controls . marginally higher in animals exhibiting more than 100% infection as compared to those with medium degree of infection (40-70%) . Monooxygenases were, therefore, analysed only in the animals registering more than 100% parasite level in the liver (Table II). The cytochrome P-450 dependent activities were significantly depressed during the infection . The decline was less prominent in aminopyrine Ndemethylase as compared to aniline hydroxylase and benzo[a]pyrene hydroxylase activities . The other microsomal marker enzymes, viz . NADH cytochrome b5 reductase and NADPH-cytochrome c reductase, did not show any significant change during the infection .

Table II Status of the hepatic microsomal cytochrome P-450 dependent monooxygenases and microsomal marker enzymes in control and L. donovani infected hamsters Enzyme

Aniline hydroxylasea Benzo[a]pyrene hydroxylaseb Aminopyrine N-demethylasea NADH-cytochrome b5 reductase ca NADPH-cytochrome e reductasea

Control

0-549 ±0-049 5 . 707 ±0-431 1 . 800 ±0-021 2-636±0-198 24 . 10 ±0-99

Infected

0 . 338 3-980 1 . 511 2-383 23 . 07

±0-022 ±0-319 ±0-019 ±0-316 ± 1 .10

% Change (P value)

- 38 . 4 - 30-2 -16 . 0 -9-5 -4-2

(0-005) (0-005) (0-05) (NS) (NS)

anmol product formed min - ' mg protein - ' ; 'relative fluorescence min - ' mg protein - ' ; Values are mean ± sD of four separate observations from separate animals. Hamsters exhibiting 100% degree of infection were included . NS, non-significant change.



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DISCUSSION Development of granulomas in liver, spleen and bone marrow are the most important histopathological features of L . donovani infection [18]. The number of granulomas in hepatic tissue has been found to increase with the increase in degree of the infection and a direct correlation has been established . Profound hepatomegaly during the infection indicates activation of proliferation of hepatic tissues . The level of microsomal protein is also maintained during the infection, indicating simultaneous proliferation of microsomal fraction . The decrease in cytodrome P-450 and haem content was directly related to the degree of infection supporting histopathological changes reported earlier during L. donovani infection [20] . However, the depression in cytochrome P-450 dependent monooxygenases was not uniform like aniline hydroxylase and benzo[a]pyrene activities showed more decrease than aminopyrine N-demethylase . Cytochrome P-450 has been shown to occur in multiple molecular forms with each species catalysing a particular type of reaction . These isoenzymic forms are genetically determined [21]. The observation, therefore, indicates a specific impairment in MFO system during leishmaniasis . Possibly the increased proliferation of hepatic tissue as well as microsomal membranes did not allow any significant change in microsomal markers, viz . NADH-cytochrome b 5 reductase and NADPH-cytochrome c reductase . Leishmania promastigotes penetrate and multiply in the Kupffer cells of hepatic tissue . Residence of the parasite in these cells may stimulate their cytolytic property and phagocytic activity. This may also be one of the possible causes of depression in MFO activities during leishmaniasis since stimulation of phagocytic activity of Kupffer cells results in secretion of certain cytolytic substances, causing impairment of MFO activities in the adjacent hepatocytes [22] . This may be the reason why the depression of MFO activities was most prominent in animals registering more than 100% infection in the liver . This derangement in the host during leishmaniasis may lead to several pharmacological, toxicological as well as physiological consequences . This would limit the ability of the host to metabolize drugs and xenobiotics, resulting in changes in efficacy and pharmacological activity of the drugs as well as adverse drug reactions . The biotransformation of several steroidal hormones through MFO system would also be affected, causing hormonal imbalance. ACKNOWLEDGEMENTS The authors are grateful to Prof. B.N . Dhawan, Director, Central Drug Research Institute, Lucknow, for providing necessary facilities . Mr . A.K . Singh is a recipient of ICMR, Junior Research Fellowship . Excellent technical assistance of Messrs S.K . Bose, O.N. Bhalla and V.K. Nigam is gratefully acknowledged . This paper is a CDRI communication No. 4414 . REFERENCES 1 . Kato R . In: Schenkman JB, Kuffer D, eds . Hepatic cytochrome P-450 monooxygenase system. Oxford : Pergamon Press, 1982 : 99 .



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