Importance of T-cell-dependent inflammatory reactions in the decline of microsomal cytochrome P450 concentration in the livers of rats infected with Fasciola hepatica

Inremorional Journal/or Parasitology, Vol. 25, No. 10, pp. 125%1262, 1995 Copyright 0 1995 Australian Society for Parasitology Elsevier Science Ltd Printed in Great Britain. All rights reserved 0020-7519(95)00052-6 002~7519/95 $9.50 + 0.00

Pergamon

RESEARCH NOTE

Importance of T-Cell-dependent Inflammatory Reactions in the Decline of Microsomal Cytochrome P450 Concentration in the Livers of Rats Infected with Fasciola hepatica FIONA

TOPFER,

LINDA M. LENTON, FYFE L. BYGRAVE CAROLYN A. BEHM*

and

Division of Biochemistry and Molecular Biology, School of Life Sciences, Australian National University, Canberra, A.C.T. 0200, Australia (Received

6 December

1994; accepted

30 May

1995)

Abstract-Topfer F., Lenton L. M., Bygrave F. L. & Behm C. A. 1995. Importance of T-cell-dependent inflammatory reactions in the decline of microsom$ cytochrome P450 concentration in the livers of rats infected with Fasciola hepatica. Znternational Journalfor Parasitology 25: 1259-1262. The concentration of cytochrome P450, measured spectiophotometrically in microsomal preparations from the livers of rata infected with 30 metacercariae of Fasciola hepatica, declined by approximately 50% at 3 weeks postinfection. Treatment of infected rats with the anti-inflammatory agent dexamethasone (2 mg/kg at 48 h intervals for 8 days prior to assay) abolished the decline in P450 content. Assay of P450 in infected congenitally athymic (nude) rats showed normal levels. These results demonstrate that the T-cell-dependent inflammatory response in the liver of the host is a necessary factor in the development of the decline in hepatic P450, which is known to compromise the metabolism of certnin drags in infected hosts. Key wor& dexamethasone;

Fusciola hepatica; liver fluke; athymic rats; inflammation.

liver

metabolism;

Infection with the liver fluke Fasciola hepatica has been shown to alter the drug-metabolizing capacity of the host’s liver in mice, rats and sheep (Somerville, Bygrave & Behm, 1995; Maffei Facino et al., 1981; Galtier, Larrieu, Tufenkji & Franc, 1986). Loss of drug-metabolizing capacity in this infection is characterized by a decline in both the total concentration of the microsomal haemoprotein cytochrome P450 and in P450-dependent enzymatic activities. In rats the loss of P450 occurs as early as 1 week after infection, with the maximal loss (approximately 50%) occurring 30 days after infection (Maffei Facino et al., 1981). The time course of P450 decline follows the same pattern as the damage to mitochon*To

whom

correspondence

should

be addressed. 1259

drug

metabolism;

trematode;

P450;

drial function that also occurs in the liver of the host during the infection. Mitochondrial dysfunction, which is characterized by uncoupling of oxidative phosphorylation (Van den Bossche, Verheyen, Verhoeven & Amouts, 1983) attenuation of absolute oxygen uptake and insensitivity to the inhibitor oligomycin, is greatest in rats at 34 weeks postinfection (Rule, Behm & Bygrave, 1989). Mitochondrial dysfunction can be prevented by treatment with the anti-inflammatory agent dexamethasone, for 1 week prior to experimentation (Hanisch et al., 1992). Dexamethasone treatment does not prevent or delay maturation of the liver flukes. We have also shown that mitochondrial dysfunction does not occur in infected athymic (nude) rats (Hanisch et al., 1992). These observations implicate the host’s inflammatory

1260

F. Topfer et al.

response in the development of the mitochondrial damage. Loss of P450 has also been observed in other parasitic infections (Tekwani, Shulka 8z Ghatak, 1988), some of which do not involve liver pathology, in viral disease (Mannering, Renton, El Azhary 8z Deloria, 1980) and in response to elevated levels of various cytokines (Ghezzi, Saccardo & Bianchi, 1986; Mannering et al., 1980; Sujita et al., 1990; Fukuda et al., 1992). In the present study the effect of treatment of infected rats with dexamethasone on the concentration of hepatic microsomal P450 was determined in order to investigate the role of the host’s inflammatory response in the decline in drug-metabolizing capacity of the liver. To assess the T-cell dependence of this phenomenon, we also tested infected athymic (nude) rats for loss of microsomal P450. Outbred male White Wistar rats from the Faculties’ Animal Care Facility, Australian National University, were kept in plastic cages in groups of 2 or 3 with commercial rat chow and water available at all times. Athymic (nude) rats (Lewis-derived nu/nu and nu/+) were obtained from the Animal Breeding Establishment, ANU. At 4-5 weeks of age the animals were infected with 30 viable metacercariae, by gastric gavage after being lightly anaesthetized with anaesthetic ether. Metacercariae of F. hepatica, maintained in the snail Lymnaea viridis, were obtained from Dr J. Boray, Elizabeth Macarthur Agricultural Research Institute, Camden Park, N.S.W. All infections were of 3 weeks duration. The Wistar rats were treated, by subcutaneous injection, with dexamethasone (2 mg/kg; Dexaphos Jurox Pty. Ltd, Riverstone, N.S.W., Australia) every 48 h for 8 days prior to being used in experiments. No treatment was given 24 h prior to experimentation. The livers of deeply anaesthetized rats (50 mg/kg Nembutal) were removed, weighed and placed in phosphate buffer [7.53 mM NazHPOd, 2.5 mM NaH2P04, 2Hz0, 1.15% (w/v) KC11 on ice. All subsequent preparative procedures were carried out at 0°C. The livers were chopped and homogenized in a Thomas C homogenizer with a motordriven Teflon pestle (10 passes). The homogenate was centrifuged for 30 min in a refrigerated RC-5B Sorvall centrifuge with an SS-34 angle rotor at 18,000 r.p.m. (39,100g). The supernatant solution was collected and further centrifuged in a Beckman L8-70M Ultracentrifuge with a SW 28 swinging bucket rotor at 27,000 r.p.m. (100,000 g) for 1 h. The pellet was collected and resuspended with a Thomas A homogenizer with hand-held Teflon pestle in phosphate buffer containing 1.15% KC1 to a final concentration between 25 and 30 mg protein/ml. Protein content was measured by the method of

control

Infected

Dexconml

Experimental

Dex-infected

Group

Fig. 1. Concentration of cytochrome P450 in the hepatic microsomal fraction of uninfected, infected, dexamethasone-treated uninfected and infected rats. Microsomes were suspended in phosphate buffer (7.53 mM Na2HP04, 2.5 mM NaH2P04 .2H20, 1.15% KCl) at approximately 2 mg protein/ml. The sample was reduced with Na dithionite and a background spectrum recorded. After gassing the sample with CO for 30 s, the difference spectrum was recorded. Dexamethasone (Dex) treatment (2 mg/kg) was administered subcutaneously every 48 h for the final 8 days of infection. All infections were of 3 weeks duration. Results are expressed as the mean+ S.E.M. (n = 4 rats in each group). Lowry, Rosebrough, Farr & Randall (1951) modified for use in a microtitre plate. The P450 content of the microsomal fraction was determined by the spectrophotometric method of Omura & Sato (1964), which measures the combined concentration of all P450 enzymes. The microsome sample was diluted to approximately 2 mg protein/ml and reduced with solid sodium dithionite: a background spectrum was recorded using a Pye Unicam PUS800 spectrophotometer between 500 and 400 nm: the sample cuvette was then gassed with carbon monoxide for 25-30 s and the difference spectrum recorded. A molar extinction coefficient of 91 cm-’ mM-’ was used to calculate the concentration of P450 (Imai & Sato, 1967). The P450 concentration in microsomes isolated from uninfected, infected, dexamethasone-treated uninfected and dexamethasone-treated infected animals is presented in Fig. 1. At 3 weeks after infection there is a marked decrease (approximately 50%) in the P450 content of liver microsomes. This is in accord with observations of other research groups (Galtier et al., 1986; Maffei Facino et al., 1981). Treatment with dexamethasone appears to prevent the loss of P450 in the infected liver as the value obtained is comparable with those obtained from uninfected-untreated and uninfected-treated animals. Although treatment with dexamethasone is known to induce some P450 enzymes, particularly the CYP3A

Importance of T-cell dependent inflammatory

IJninfecrodNude

InfcctedNide

Experimental

-=-Y&w=

Group

Fig. 2. Concentration of hepatic microsomal cytochrome P450 in infected and uninfected nude (nu/nu) and infected heterozygote (nu/+) rats. Microsomes were prepared and P450 content assayed as described in the text and in the legend to Fig. 1. All infections were of 3 weeks duration. Results are expressed as the mean of 2 rats in eachgroup; symbols represent the values for individual rats. family, this was not evident in the treatment and assay protocols used here. The P450 concentration in microsomal preparations from control and infected nude (nu/nu) and

infected heterozygote (nu/+) rats is shown in Fig. 2. It is clear that the heterozygotes exhibited similar loss of P450 to the outbred Wistar rats and that nude infected rats possessedlevels of P450 similar to the uninfected rats. Dexamethasone is a glucocorticoid that is antiinflammatory because it kills T-lymphocytes and inhibits eicosanoid, cytokine and nitric oxide production as well as leucocyte adhesion during the inflammatory response (e.g. Alnemri & Litwack, 1989; Cidlowski, 1982; Rolfe, Hughes, Armour & Sewell, 1992; Moncada & Palmer, 1991; Cronstein et al., 1992). We have reported previously that treatment of infected rats with dexamethasone reduces the macroscopic infiltration of leukocytes into liver fluke tracks in the liver (Hanisch et al., 1992). As treatment with dexamethasone prevented the loss of P450 during infection (Fig. l), it is likely that, in rats, factors associated with the inflammatory response are involved in the induction of this lesion. The results from the infection of nude rats (Fig. 2) confirm that T-cells are necessary for the development of this biochemical lesion. Interactions between components

response and hepatic P450 concentration are known from other pathological conditions. Various cytokines and effector molecules have been reported to induce a decrease in the concentration or activity of hepatic

microsomal

P450,

including

interleukin-1

(Sujita et al., 1990), interleukin-6 (Fukuda et

al.,

1261

1992), interferon y (Mannering et al., 1980), tumour necrosis factor (Ghezzi et al., 1986), and nitric oxide (Wink et al., 1993; Khatsenko, Gross, Rifkind & Vane, 1993). It is also possible that the prostaglandins may be involved in the loss of P450 through indirect activation of protein kinases involved in P450 degradation (Taniguchi, Pyerin & Stier, 1985). Hepatic oxidative stress is at least a partial factor contributing to the loss of P450 in fluke-infected rats (Maffei Facino et al., 1989, 1993). The mechanisms leading to the loss of hepatic P450 in fluke-infected rats appear to be selective, at least in part, for different isofoims of P450, as it has been reported (Galtier, Larrieu & Beaune, 1986) that the constitutive CYP2CI 1 enzyme declines whereas the inducible CYP2Bl and CYPlAl enzymes do not. Such severe effects of liver fluke infection on the potential capacity of the host to metabolize drugs are important when considering anthelmintic treatments, because treatment with certain anti-parasitic drugs or pro-drugs may be less effective or more toxic to the host as a result of the decrease in drug-metabolizing capacity. AcknowledgementsWe would like to thank Dr J. C. Boray for supply of metacercariae, Dr D. A. Day for use of the Pye Unicam spectrophotometer, Dr G. D. Buflinton for advice and the Australian Research Council for partial financial support of this work. REFERENCES

Alnemrie S. & Litwack G. 1989. Glucocorticoid-induced lymphocytolysis is not mediated by an induced endonuclease. Journal of Biological Chemistry 264: 41044111. Cidlowski J. A. 1982. Glucocorticoids stimulate ribonucleic acid degradation in isolated rat thymic lymphocytes in vitro. Endrocrinology 111: 184190. Cronstein B. N., Kimmel S. C., Levin R. I., Martiniuk F. & Weissmann G. 1992. A mechanism for the antiinflammatory effects of corticosteroids-the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule-l and intercellular adhesion molecule-l. Proceedings of the National Academy of Sciences of the United States of America 89: 9991-9995. Fukuda Y., Ishida N., Noguchi T., Kappas A. 62 Sassa S. 1992. Interleukin-6 down regulates the expression of transcripts encoding cytochrome P450 IAl, IA2 and IIIA3 in human hepatoma cells. Biochemical and Biophysical

of the immune

reactions

Research

Communications

184: 96&965.

Galtier P., Larrieu G. & Beaune P. 1986. Characterization of the microsomal cytochrome P-450 species inhibited in rat liver in the course of fascioliasis. Biochemical Pharmacology

35: 434H347.

Galtier P., Larrieu G., Tufenkji A. E. & Franc M. 1986. Incidence of experimental fascioliasis on the activity of drug-metabolizing enzymes in lamb liver. Drug Metabolism and Disposition 14: 137-141.

1262

F. Topfer

Ghezzi P., Saccardo B. & Bianchi M. 1986. Recombinant tumour necrosis factor depresses cytochrome P450dependent microsomal drug metabolism in mice. Biochemical and Biophysical Research Communications 136: 316321. Han&h M. J. E., Topfer F., Lenton L. M., Behm C. A. & Bygrave F. L. 1992. Restoration of mitochoadrial energy-linked reactions following dexamethasoae treatment of rats infected with the liver fluke Fasciola hepatica. Biochimica et Biophysics Acta 1139: 196202. Imai Y. & Sato R. 1967. Conversion of P-450 to P-420 by neutral salts and some other reagents. European Journal of Biochemistry 1: 419426. Khatseako 0. G., Gross S. S., Rifkind A. B. & Vane J. R. 1993. Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulaats. Proceedings of the National Academy of Sciences of the United States of America 90: 11,147-11,151. Lowry 0. H., Rosebrough N. J., Farr A. L. & Randall R. I. 1951. Protein measurement with the Folin reagent. Journal of Biological Chemistry 193: 257-275. Maffei Facino R., Carini M., Aldini G., Ceserani R., Casciarri I., Cavalletti E. & Verderio L. 1993. Efficacy of glutathione for treatment of fascioliasis-an investigation in the experimentally infested rat. ArzneimitteZForschunglDrug Research 43-l: 45S460. Maffei Faciao R., Carini M., Bertuletti R., Genchi C. & Malchiodi A. 1981. Decrease of the in vitro drug-metabolizing activity of the hepatic mixed function oxidase system in rats infected experimentally with Fasciola hepatica: pharmacological implications. Pharmacological Research Communications 13: 731-742. Maffei Faciao R., Cariai M., Geachi C., Tofaaetti 0. & Casciarri I. 1989. Participation of lipid peroxidation in the loss of hepatic drug-metabolizing activities in experimental fascioliasis in the rat. Pharmacology Research 21(5): 549-560. Maaaering G. J., Renton K. W., El Azhary R. & Deloria L. B. 1980. Effects of interferon-inducing agents on hepatic cytochrome P-450 drug metabolizing systems. Annals of the New York Academy of Sciences 350: 314331.

et al. Moncada S. & Palmer R. M. J. 1991. Inhibition of the induction of nitric oxide synthase by glucocorticoids: yet another explanation for their anti-inflammatory effects? Trends in Pharmacological Sciences 12: 13&131. Omura T. & Sato R. 1964. The carbon monoxide-binding pigment of liver microsomes I. Evidence for its hemoprotein nature. Journal of Biological Chemistry 239: 237G-2378. Rolfe F. G., Hughes J. M., Armour C. L. & Sewell W. A. 1992. Inhibition of iaterleukia-5 gene expression by dexamethasone. Immunology 77: 494499. Rule C. J., Behm C. A. & Bygrave F. L. 1989. Aberrant energy linked reactions in mitochondria isolated from the livers of rat infected with the liver fluke Fasciola hepatica. Biochemical Journal 260: 517-523. Somerville A. C., Bygrave F. L. & Behm C. A. A study of hepatic mitochoadrial respiration and microsomal cytochrome Pd5s content in mice infected with the liver fluke Fasciola hepatica. International Journal for Parasitology 26: 667-672. Sujita K., Okuno F., Tanaka Y., Hirano Y., Iaamoto Y., Eto S. & Arai M. 1990. Effects of interleukia 1 (IL-l) on the levels of cytochrome P-450: involving IL-l receptor on the isolated hepatocytes of rat. Biochemical and Biophysical Research Communications 168: 1217-1222. Taaiguchi H., Pyerin W. & Stier A. 1985. Conversion of hepatic microsomal cytochrome P-450 to P-420 upon phosphorylation by cyclic AMP dependent protein kiaase. Biochemical Pharmacology 34: 183551837. Tekwaai B. L., Shulka 0. P. & Ghatak S. 1988. Altered drug metabolism in parasitic diseases. Parasitology Today 4: 4-10. Van den Bossche H., Verheyea A., Verhoeven H. & Arnouts D. 1983. Alterations in rat liver mitochondria caused by Fasciola hepatica. In: From Parasitic Infection to Parasitic Disease (Edited by Gigase P. L. & Van Marck E. A. E.), pp. 3(r38. S. Karger, Basel. Wink D. A., Osawa Y., Darbyshire J. F., Jones C. R., Eshenaur S. C. & Nims R. W. 1993. Inhibition of cytochromes-P450 by nitric oxide and a nitric oxidereleasing agent. Archives of Biochemistry and Biophysics 300: 115-123.