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Modulation of Hepatic Cytochrome P450 during Listeria Monocytogenes Infection of the Brain
Modulation of Hepatic Cytochrome P450 during Listeria Monocytogenes Infection of the Brain ELENA GARCIA DEL BUSTO CANO, KENNETH W. RENTON Department of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
Received 11 December 2002; revised 26 February 2003; accepted 11 March 2003
ABSTRACT: Hepatic cytochrome P450 enzymes can be modulated during systemic infections. Inflammatory responses in the brain have also been shown to cause a significant decrease in the levels and activities of important cytochrome P450 isoforms in the liver. We determined some of the effects of central nervous system (CNS) Listeria monocytogenes infection on hepatic cytochrome P450 systems in rats. Intracerebroventricular injection of L. monocytogenes resulted in a time-dependent modulation of CYP1A, CYP2B, and CYP3A activities in the liver. Total hepatic cytochrome P450 content was significantly lowered 48 h after administration of the bacterium, and hepatic CYP1A and CYP2B activities were significantly altered 48 and 72 h after infection, respectively, whereas CYP3A activity and protein content were depressed 72 h after the insult. Bacterial load in the brain increased dramatically over a 72-h period, but the number of bacteria cultured from liver over this time period was relatively small. Therefore, an infection largely confined to the CNS in the rat results in abnormal activity levels of certain hepatic cytochrome P450 enzymes crucial in drug metabolism. If such a response also occurs in humans, this has the potential to produce serious complications with drug and endogenous substrate metabolism in patients with an infectious disease involving the CNS. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1860–1868, 2003
Keywords:
cytochrome P450; meningitis; Listeria monocytogenes; drug metabolism
INTRODUCTION Generalized inflammation and viral, bacterial, and parasitic infections have widely been shown to modulate cytochrome P450 expression and activity in the liver, lung, intestine, kidney, and other organs of several species including humans.1–5 In most but not all cases, this alteration is manifested as a depression in protein content via pretranslational mechanisms, and results
Correspondence to: Kenneth W. Renton (Telephone: 902494-2562; Fax: 902-494-1388; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 1860–1868 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
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in altered drug and endogenous substrate metabolism.1,5 The elimination of a number of drugs (e.g., theophylline, caffeine, carbamazepine, warfarin, quinine, dextromethorphan) in humans during periods of infectious disease has been compromised and resulted in serious drug toxicity.5 During lipopolysaccharide (LPS)-evoked inflammatory responses that are largely confined to the central nervous system (CNS), there is a loss in drug biotransformation capacity not only in the brain but also in peripheral organs such as the liver.6,7 Even though no inflammatory response could be observed in the liver, a decrease in total cytochrome P450 content and CYP2C11, CYP1A, CYP2B, CYP2E1, and CYP3A activities and/or contents in that organ was observed after the injection of LPS into the lateral ventricle of the
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brain.6,7 These authors suggest that LPS-evoked inflammation within the brain modifies cytochrome P450-dependent reactions within that organ7 and also evokes a signaling mechanism that results in a concomitant decline in drug biotransformation in the liver.6,7 Listeria monocytogenes is a gram-positive, nonspore forming bacterium8 often involved in community acquired bacterial meningitis.8,9 Infection of the CNS by L. monocytogenes accounts for about 5% of all bacterial CNS infections in the United States and is complicated by its intra- and extracellular distribution.8,10–12 L. monocytogenes has an opportunistic nature, which is revealed in its preference toward newborns, pregnant women, the elderly, and immunocompromised individuals, although it can sometimes infect healthy persons.8,13–15 This predilection is reflected in the fact that CNS listeriosis is one of the most frequent forms of listeriosis in newborns and immunocompromised individuals.11,15 Furthermore, the appearance of human immunodeficiency virus, as well as an increase in bone marrow and organ transplantations, has provided Listeria with a greater number of individuals at risk,8,13,15 many of which are receiving treatment for a wide variety of conditions. A study by Jurado et al.16 reported the estimated incidence of L. monocytogenes meningitis and bacteremia among human immunodeficiency virus-infected patients and patients with acquired immunodeficiency syndrome to be 65–145 times higher than in the general population, with a mortality of 29%. The organism is also often encountered in cancer patients.14 In addition, listerial rhombencephalitis often occurs in sheep and cattle.11,15 Administration of L. monocytogenes directly into the CNS as used in the current animal model is a well established model that mimics human L. monocytogenes infection of the CNS.10 The bacterium infects the ependymal cells lining the ventricles first, and then expands to the parenchyma of the brain where it shows predilection for macrophages before extending the infection to neurons. This process culminates with destruction of these cells, which results in brain stem encephalitis similar to that observed in human cerebral listeriosis.10 The present study suggests that a loss in the capacity to metabolize drugs may also occur during CNS infection with L. monocytogenes which has the potential to alter drug therapy particularly in high-risk human and veterinary populations who receive a large number of drugs.
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Previously, systemic infection with L. monocytogenes has been reported to decrease cytochrome P450 and drug elimination in the livers of rodents.17–19 It is unknown if drug disposition is altered during episodes of bacterial meningitis. The purpose of this study was to evaluate the levels of common cytochrome P450 forms and their activities (CYP1A, CYP2B, and CYP3A) in the liver during host defense responses to an infection with L. monocytogenes within the brain.
METHODS Chemicals Brain Heart Infusion Broth (BHI) was purchased from Becton Dickinson Microbiology Systems (Cockeysville, MD). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Frozen aliquots of L. monocytogenes (serotype 4b) were provided by Dr. Rafael Gardun˜o (Microbiology Department, Dalhousie University). The 5% Trypsin Soy Agar blood plates used for bacterial culture were purchased from the Microbiology Laboratory at the Queen Elizabeth II Health Sciences Centre (Halifax, Nova Scotia). The anti-rat CYP3A1/2 monoclonal antibody (no. 2-13-1) was kindly provided by Dr. H. Gelboin (National Cancer Institute, Bethesda, MD) and the anti-rat CYP1A1/2 and CYPB11/2 polyclonal antibodies were purchased from Gentest Corp. (Woburn, MA). Animals and Treatment All experiments were performed in compliance with Dalhousie University regulations. Male Sprague-Dawley rats (175–200 g) were obtained from Charles River Labs (Quebec, Canada) and housed in groups of two or three per cage on commercially available standard wood chips in a 12-h light/dark cycle. Rats were allowed to acclimatize for 1 week before experimental use, during which they were fed Standard Purina Rat Chow and water ad libitum. Animals were anesthetized with an ip injection of 0.2 mL of a freshly prepared anesthetic cocktail (55% ketamine, 28% xylazine, 11% acepromazine). L. monocytogenes was injected directly into the lateral ventricle of the brain using a KOPF stereotaxic frame (Tujunga, CA) with coordinates: þ1.5-mm lateral from bregma and 4.7-mm below the skull surface. Animals were allowed free access to food and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
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water from this time until killed at 24, 48, or 72 h after bacterial administration.
L. Monocytogenes Inoculum Preparation and Bacterial Burden Determination For each experiment, a Listeria aliquot was thawed and plated onto a 5% Trypsin Soy Agar blood plate, followed by an overnight incubation at 378C. Isolated bacterial colonies were then selected, inoculated into 4 mL of sterile BHI, and incubated at 378C with vigorous shaking for 12 h. After this incubation period, 50 mL were placed into 5 mL of fresh BHI broth and incubated for 3 h at 378C with shaking, to reach the exponential phase of bacterial growth. Bacterial growth and density of the solution were then determined by an absorbance determination of a 1:10 dilution of the broth using a wavelength of 620 nm. An absorbance of 0.6 corresponds to a solution containing 5 108 colony forming units (CFU) of bacteria/milliliter.17 Rats were injected icv with 5 102 CFU of L. monocytogenes in a total volume of 10 mL and control animals received an equal volume of 0.9% sterile saline. Bacterial counts in tissues were used to define the extent of infection at the time rats were killed. Liver and brain samples were weighed and homogenized in 5 mL of sterile phosphate buffered saline under sterile conditions. Dilutions of the homogenates were then made in sterile 0.9% NaCl and plated onto blood agar plates. The plates were incubated at 378C for 24 h and the number of colonies grown was counted at specific dilutions. Bacterial count was expressed as CFU/milligram of tissue.
Tissue Preparation Hepatic microsomes were prepared by the method described by el-Masry et al.20 Animals were killed 24, 48, or 72 h post-treatment by CO2 asphyxiation; livers were rinsed with cold 1.15% KCl and homogenized in 20 mL of the same solution with a Polytron homogenizer. The homogenates were centrifuged at 10,000g for 10 min at 48C and the supernatants were re-centrifuged at 110,000g for 40 min. The resulting pellets were resuspended in 2.5 mL of glycerol buffer (50 mM monobasic KH2PO4, pH 7.5, 20% glycerol, 0.40% KCl) using a glass homogenizer. The suspension was divided into 750-mL aliquots and stored at 708C. Microsomal protein content was determined by the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
method of Lowry et al.21 using bovine serum albumin as a standard. Cytochrome P450 Activities CYP1A1/2 activity of liver microsomes was determined by the formation of resorufin from ethoxyresorufin and CYP2B1/2B2 activity was determined by measuring the formation of resorufin from benzyloxyresorufin.22 CYP3A2 activity of hepatic microsomes was determined by the formation of 4-OH midazolam (MDZ) from MDZ detected by high-performance liquid chromatography.23–25 Reaction mixtures contained 50 mM phosphate buffer, 1 mM NADPH, 0.5 mg of microsomal protein/milliliter, and 12.5 mM MDZ. Formation of the 4-OH MDZ metabolite was linear for up to 5 min and a microsomal protein concentration of 2 mg/mL. Reactions were stopped with 5 mL of methylene chloride and the internal standard, phenacetin, was added. The samples were mixed, centrifuged, and the organic phase was transferred to glass tubes to be evaporated under a nitrogen stream. Samples were resuspended in 125 mL of mobile phase (29% methanol: 28% acetonitrile: 41% 0.01 M KH2PO4, pH 7.4: 2% tetrahydrofuran). A volume of 50 mL of resuspended product was injected on to a Beckman C8 column (5 mm 4.5 mm 25 mm i.d.) using a UV detector at 220 nm to detect the metabolite. All activities were normalized to the amount of protein in each sample. Hepatic P450 Content Determination Hepatic microsomal total cytochrome P450 content was determined as described by the method of Omura and Sato26 using a molar extinction coefficient for the reduced P450-CO complex of 91 cm1 mM1. Immunohistochemistry Twenty-four and 48 h after icv injection of L. monocytogenes or saline, rats were anesthetized with approximately 70 mg/kg pentobarbital and perfused transcardially with 60 mL of 0.9% saline followed by about 120 mL of cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4). Whole brains were carefully removed and placed into vials containing paraformaldehyde for 24 h, followed by cryoprotection in 30% sucrose at 48C until completely submerged. Brains were then sectioned into 40-mm coronal sections with a
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freezing microtome and stored at 48C in Millonig’s solution until further processing. Heat shock protein 27 (hsp27) staining was performed on the sections according to the protocol described by Garcion et al.27 using a rabbit antimouse hsp25 polyclonal antibody (1:5000 dilution in 2% goat serum) (StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) This antibody cross-reacts with the rat hsp27. The secondary antibody was biotinylated goat antirabbit immunoglobulin (Ig)G antibody (1:400 dilution in PB) detected with Avidin Biotin solution, followed by exposure to 3,30 -diaminobenzidine tetrahydrochloride. Western Blot Analysis Each lane was loaded with a 10-mL volume containing 25 mg of microsomal protein. Samples were separated on a 7.5% Tris-HCl Ready Gel (Bio-Rad Labs, Hercules, CA) under nonreducing conditions. The proteins were then transferred to an Immobilon P membrane using a wet transfer. CYP3A1/2 was detected using a mouse anti-rat CYP3A1/2 monoclonal antibody. Membranes were blocked for 2 h at room temperature (RT) in 5% skim milk, followed by incubation with the primary antibody (1:5000 dilution) in 2% skim milk for 1 h at RT. A peroxidase-conjugated anti-mouse IgG secondary antibody was used to proceed with the immunological detection of the protein using standard procedures. CYP1A1/2 and CYP2B1/2 were detected using goat anti-rat CYP1A1/2 and CYP2B1/2 polyclonal antibodies, respectively. Membranes were blocked for 2 h at RT in 5% skim milk containing 0.5% bovine serum albumin, followed by incubation with the primary antibody (1:500 dilution) in 2.5% skim milk for 1 h at RT. The secondary antibody used was an anti-goat IgG conjugated to peroxidase. Bands were visualized using Supersignal Ultra chemiluminescent substrate (Pierce, Rockford, IL). Blots were exposed to X-ray film and band intensities measured using the program Molecular AnalystTM (Bio-Rad, Mississauga, Ontario, Canada). A set of Kaleidoscope prestained molecular weight standards (Bio-Rad, Mississauga, Ontario, Canada) was used for all blots to determine the size of the bands detected. Statistical Analysis Comparisons between groups were performed using two-way analysis of variance, followed by
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Bonferroni post-tests16 in all figures. A p value < 0.05 was considered significant. Error bars on all graphs represent the standard error of the mean of that group.
RESULTS L. Monocytogenes Infection and Bacterial Load in Brain and Liver A dose of 5 102 CFU of L. monocytogenes injected into the lateral ventricle of the brain produced a nonlethal infection, and no severe morbidity was observed throughout the duration of these experiments. The rats injected with L. monocytogenes, however, had signs of lethargy, decreased social interaction, decreased grooming, and a slight weight loss. The time course of the infection was monitored by measuring the number of bacteria present in the brain and liver during the first 72 h after administration of the organism. The number of CFU of bacteria found per milligram of tissue in the brain increased rapidly from 3.6 103 CFU at 24 h, 3.7 104 CFU at 48 h, and reached over 4 104 CFU by 72 h. Only a small number of bacteria were detected per milligram of liver at 24 h (4 10 CFU) and 48 h (1.7 102 CFU) after Listeria infection and this increased to 1.3 103 CFU at 72 h. No bacteria were detected in control animals that received saline. Expression of hsp27 in the Brain after L. Monocytogenes Infection The development of an inflammatory response to infection in the brain was performed by assessing immunohistochemical staining of coronal brain sections with an antibody against hsp27. Immunoreactivity to hsp27 was expressed in the areas surrounding the ventricles and in the parenchyma next to these regions at 24 and 48 h after infection (Figs. 1 and 2, respectively). Immunoreactivity in the corresponding sections taken from control animals treated with saline was low. Effect of CNS Listeriosis on CYP1A, CYP2B, and CYP3A At 48 h after infection, the ethoxyresorufin deakylase activity (CYP1A) was increased by 80% but remained unchanged at 24 and 72 h (Fig. 3a). Benzyloxyrexorufin de-alkalase (CYP2B) and MDZ hydroxylase activity (CYP3A) remained JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
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Figure 1. Expression of hsp27 around the lateral (A and B) and third (C and D) ventricles 24 h after icv administration of 5 102 CFU of L. monocytogenes. Panels A and C correspond to brain sections from control animals, whereas panels B and D are the counterparts from L. monocytogenes infected animals. All images were taken at an original magnification of 5.
unchanged for 24 and 48 h after infection before decreasing at 72 h (Fig. 3b,c). No changes in CYP1A1/2 or CYP2B1/2 protein content were observed at any of the time points studied as reflected by Western blot analysis (Fig. 4a,b) whereas CYP3A1/2 content was significantly decreased at 72 h after L. monocytogenes administration (Fig. 4c). A decrease in total P450 levels in the liver was observed after a 48-h infection period (Fig. 5).
DISCUSSION It has now been established that systemic inflammation and infections can trigger a mechanism resulting in a direct or indirect alteration in cytochrome P450 forms and their corresponding activities.1,5 This results in changes in the capacity of the liver to metabolize drugs and chemicals and has a profound effect on the pharmacokinetics of certain drugs. Hepatic P450 regulation also seems to be compromised during inflammaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
tory responses originating in the brain.5–7 The mechanism responsible for the signaling between the CNS and the periphery is complicated and may involve the expression of cytokines, prostaglandins, and other mediators.5 The current study suggests that changes to drug biotransformation in the liver also occur during bacterial infections that are largely confined to the CNS. Infection of the brain by L. monocytogenes produced changes in a number of cytochrome P450 forms of the enzyme in the liver. The infection was confirmed by the recovery of bacteria from the brain stem of the infected rats 24, 48, and 72 h after icv administration, and a high immunoreactivity against hsp27 in the brains of infected animals. In contrast, the number of bacteria found in the liver at these times was small, indicating that a generalized systemic infection did not occur. In terms of the activities of CYP450 in the liver, different effects were observed at different points after icv injection, which may be an indication of the stage of development of infection. CYP1A activity shows a strong though temporary increase, 48 h after
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Figure 2. Expression of hsp27 around the lateral (A and B) and third (C and D) ventricles 48 h after icv administration of 5 102 CFU of L. monocytogenes. Panels A and C correspond to brain sections from control animals, whereas B and D are the counterparts from L. monocytogenes infected animals. All images were taken at an original magnification of 5.
infection, which returned to control levels by 72 h. This pattern is similar to that described for a L. monocytogenes systemic infection in mice by Armstrong and Renton,19 who showed a significant increase in CYP1A mRNA and protein at 12 and 24 h, followed by a profound loss in enzyme during the period when the bacterial level in the liver was very high. The delayed onset in our study (i.e., 48 vs. 12 h) is likely attributed to the time required for the infection to develop and produce signaling molecules responsible for the hepatic loss. Both CYP2B and CYP3A activities were significantly reduced 72 h after Listeria administration, but only CYP3A microsomal protein was altered. Although the bacterial load in the liver was relatively small at this time, it had increased to more than 1 103 CFU/mg liver and it is possible that the response to the increasing number of organisms was responsible for the loss in CYP3A at this time. The apparent discrepancies between the protein levels for specific cytochrome P450 forms and the corresponding enzyme activities observed at certain times in these experiments is difficult to
explain but similar discrepancies for several forms of cytochrome P450 have previously been reported in a number of studies using LPS and a Staphylococcal enterotoxin.28–30
CONCLUSIONS These experiments show for the first time that a live bacterial infection largely confined to the CNS modulates cytochrome P450 enzymes in the liver of the rat. The loss of enzyme in the liver is time and bacterial load dependent, which reflects the dynamic nature not only of disease, but also of the concomitant regulation of cytochrome P450 enzymes that follows. The observation that total hepatic cytochrome P450 content was significantly lowered 48 h after infection implies that other isoforms, besides those we analyzed, may be affected by this condition. If this occurs in humans, the implications of these findings to the medical community are important. The number of drugs known to be metabolized by these JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
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Figure 3. Effect of CNS infection by L. monocytogenes on hepatic CYP1A (a), CYP2B (b), and CYP3A (c) activities. Male Sprague-Dawley rats received a single icv injection of 5 102 CFU of L. monocytogenes contained in a total volume of 10 mL of sterile 0.9% NaCl. Control animals received 10 mL of 0.9% NaCl. Rats were killed 24, 48, and 72 h after infection. Values are reported as percent of control SEM (n ¼ 8 for 24 h, nsaline ¼ 8, nlisteria ¼ 9 for 48 h, and n ¼ 4 for 72 h). ***Significantly different from control levels, p < 0.001; **Significantly different from control levels, p < 0.01; *Significantly different from control levels, p < 0.05. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
Figure 4. Effect of CNS infection by L. monocytogenes on hepatic protein content of CYP1A (a), CYP2B (b), and CYP3A (c). Treatment and groups were identical to that described in Figure 3. **Significantly different from control values, p < 0.01.
enzymes is numerous and may lead to an unpredictability of drug metabolism in individuals with a CNS bacterial infection. Because a significant proportion of the population is at risk for CNS listeriosis, the number of potential drug
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7.
8.
9. Figure 5. Effect of CNS infection by L. monocytogenes on total hepatic P450 content. Treatment and groups were identical to that described in Figure 3. *Significantly different from control levels, p < 0.05.
toxicities in these individuals could be a serious problem in therapeutics.
ACKNOWLEDGMENTS The authors thank Rafael and Elizabeth Gardun˜o for kindly providing the L. monocytogenes strain, as well as their expertise and patience. E. Garcia del Busto Cano was a recipient of a Killam Trusts studentship. This work was supported by the Canadian Institute of Health Research (CIHR). A portion of this work was presented in abstract form at the ASBMB/ASPET/FPS/PSC 2000 joint meeting in Boston, MA.
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Differential alterations in levels of hepatic microsomal P450 isozymes following intracerebroventricular injection of bacterial lipopolysaccharide in rats. Arch Toxicol 72:492–498. Renton KW, Nicholson TE. 2000. Hepatic and central nervous system cytochrome P450 are down-regulated during lipopolysaccharide-evoked localized inflammation in the brain. J Pharmacol Exp Ther 294:524–530. Mylonakis E, Hohmann EL, Calderwood SB. 1998. Central nervous system infection with Listeria monocytogenes: 33 years’ experience at a general hospital and review of 776 episodes from the literature. Medicine 77:313–336. Taege AJ. 1999. Listeriosis: Recognizing it, treating it, preventing it. Cleve Clin J Med 66:375–380. Schluter D, Buck C, Reiter S, Meyer T, Hof H, Deckert-Schluter M. 1999. Immune reactions to Listeria monocytogenes in the brain. Immunobiology 201:188–195. Altimira J, Prats N, Lopez S, Domingo M, Briones V, Dominguez L, Marco A. 1999. Repeated oral dosing with Listeria monocytogenes in mice as a model of central nervous system listeriosis in man. J Comp Pathol 121:117–125. Schluter D, Chahoud S, Lassmann H, Schumann A, Hof H, Deckert-Schluter M. 1996. Intracerebral targets and immunomodulation of murine Listeria monocytogenes meningoencephalitis. J Neuropathol Exp Neurol 55:14–24. Jurado RL, Farley MM, Pereira E, Harvey RC, Schuchat A, Wenger JD, Stephens DS. 1993. Increased risk of meningitis and bacteremia due to -Listeria monocytogenes in patients with human immunodeficiency virus infection. Clin Infect Dis 17:224–227. Carpentier AF, Bernard L, Poisson M, Delattre JY. 1996. Central nervous system infections in patients with malignant diseases. Rev Neurol (Paris) 152: 587–601. Va´zquez-Boland JA, Kuhn M, Berche P, Chakraborty T, Domı´nguez-Bernal G, Goebel W, Gonza´lez-Zorn B, Wehland J, Kreft J. 2001. Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14:584– 640. Miller RG. 1981. Simultaneous statistical inference, 2nd ed. New York: Springer Verlag. Azri S, Renton KW. 1987. Depression of murine hepatic mixed function oxidase during infection with Listeria monocytogenes. J Pharmacol Exp Ther 243:1089–1094. Azri S, Renton KW. 1991. Factors involved in the depression of hepatic mixed function oxidase during infections with Listeria monocytogenes. Int J Immunopharmacol 13:197–204. Armstrong SG, Renton KW. 1993. Mechanism of hepatic cytochrome P450 modulation during
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Received 11 December 2002; revised 26 February 2003; accepted 11 March 2003
ABSTRACT: Hepatic cytochrome P450 enzymes can be modulated during systemic infections. Inflammatory responses in the brain have also been shown to cause a significant decrease in the levels and activities of important cytochrome P450 isoforms in the liver. We determined some of the effects of central nervous system (CNS) Listeria monocytogenes infection on hepatic cytochrome P450 systems in rats. Intracerebroventricular injection of L. monocytogenes resulted in a time-dependent modulation of CYP1A, CYP2B, and CYP3A activities in the liver. Total hepatic cytochrome P450 content was significantly lowered 48 h after administration of the bacterium, and hepatic CYP1A and CYP2B activities were significantly altered 48 and 72 h after infection, respectively, whereas CYP3A activity and protein content were depressed 72 h after the insult. Bacterial load in the brain increased dramatically over a 72-h period, but the number of bacteria cultured from liver over this time period was relatively small. Therefore, an infection largely confined to the CNS in the rat results in abnormal activity levels of certain hepatic cytochrome P450 enzymes crucial in drug metabolism. If such a response also occurs in humans, this has the potential to produce serious complications with drug and endogenous substrate metabolism in patients with an infectious disease involving the CNS. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1860–1868, 2003
Keywords:
cytochrome P450; meningitis; Listeria monocytogenes; drug metabolism
INTRODUCTION Generalized inflammation and viral, bacterial, and parasitic infections have widely been shown to modulate cytochrome P450 expression and activity in the liver, lung, intestine, kidney, and other organs of several species including humans.1–5 In most but not all cases, this alteration is manifested as a depression in protein content via pretranslational mechanisms, and results
Correspondence to: Kenneth W. Renton (Telephone: 902494-2562; Fax: 902-494-1388; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 1860–1868 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
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in altered drug and endogenous substrate metabolism.1,5 The elimination of a number of drugs (e.g., theophylline, caffeine, carbamazepine, warfarin, quinine, dextromethorphan) in humans during periods of infectious disease has been compromised and resulted in serious drug toxicity.5 During lipopolysaccharide (LPS)-evoked inflammatory responses that are largely confined to the central nervous system (CNS), there is a loss in drug biotransformation capacity not only in the brain but also in peripheral organs such as the liver.6,7 Even though no inflammatory response could be observed in the liver, a decrease in total cytochrome P450 content and CYP2C11, CYP1A, CYP2B, CYP2E1, and CYP3A activities and/or contents in that organ was observed after the injection of LPS into the lateral ventricle of the
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brain.6,7 These authors suggest that LPS-evoked inflammation within the brain modifies cytochrome P450-dependent reactions within that organ7 and also evokes a signaling mechanism that results in a concomitant decline in drug biotransformation in the liver.6,7 Listeria monocytogenes is a gram-positive, nonspore forming bacterium8 often involved in community acquired bacterial meningitis.8,9 Infection of the CNS by L. monocytogenes accounts for about 5% of all bacterial CNS infections in the United States and is complicated by its intra- and extracellular distribution.8,10–12 L. monocytogenes has an opportunistic nature, which is revealed in its preference toward newborns, pregnant women, the elderly, and immunocompromised individuals, although it can sometimes infect healthy persons.8,13–15 This predilection is reflected in the fact that CNS listeriosis is one of the most frequent forms of listeriosis in newborns and immunocompromised individuals.11,15 Furthermore, the appearance of human immunodeficiency virus, as well as an increase in bone marrow and organ transplantations, has provided Listeria with a greater number of individuals at risk,8,13,15 many of which are receiving treatment for a wide variety of conditions. A study by Jurado et al.16 reported the estimated incidence of L. monocytogenes meningitis and bacteremia among human immunodeficiency virus-infected patients and patients with acquired immunodeficiency syndrome to be 65–145 times higher than in the general population, with a mortality of 29%. The organism is also often encountered in cancer patients.14 In addition, listerial rhombencephalitis often occurs in sheep and cattle.11,15 Administration of L. monocytogenes directly into the CNS as used in the current animal model is a well established model that mimics human L. monocytogenes infection of the CNS.10 The bacterium infects the ependymal cells lining the ventricles first, and then expands to the parenchyma of the brain where it shows predilection for macrophages before extending the infection to neurons. This process culminates with destruction of these cells, which results in brain stem encephalitis similar to that observed in human cerebral listeriosis.10 The present study suggests that a loss in the capacity to metabolize drugs may also occur during CNS infection with L. monocytogenes which has the potential to alter drug therapy particularly in high-risk human and veterinary populations who receive a large number of drugs.
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Previously, systemic infection with L. monocytogenes has been reported to decrease cytochrome P450 and drug elimination in the livers of rodents.17–19 It is unknown if drug disposition is altered during episodes of bacterial meningitis. The purpose of this study was to evaluate the levels of common cytochrome P450 forms and their activities (CYP1A, CYP2B, and CYP3A) in the liver during host defense responses to an infection with L. monocytogenes within the brain.
METHODS Chemicals Brain Heart Infusion Broth (BHI) was purchased from Becton Dickinson Microbiology Systems (Cockeysville, MD). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Frozen aliquots of L. monocytogenes (serotype 4b) were provided by Dr. Rafael Gardun˜o (Microbiology Department, Dalhousie University). The 5% Trypsin Soy Agar blood plates used for bacterial culture were purchased from the Microbiology Laboratory at the Queen Elizabeth II Health Sciences Centre (Halifax, Nova Scotia). The anti-rat CYP3A1/2 monoclonal antibody (no. 2-13-1) was kindly provided by Dr. H. Gelboin (National Cancer Institute, Bethesda, MD) and the anti-rat CYP1A1/2 and CYPB11/2 polyclonal antibodies were purchased from Gentest Corp. (Woburn, MA). Animals and Treatment All experiments were performed in compliance with Dalhousie University regulations. Male Sprague-Dawley rats (175–200 g) were obtained from Charles River Labs (Quebec, Canada) and housed in groups of two or three per cage on commercially available standard wood chips in a 12-h light/dark cycle. Rats were allowed to acclimatize for 1 week before experimental use, during which they were fed Standard Purina Rat Chow and water ad libitum. Animals were anesthetized with an ip injection of 0.2 mL of a freshly prepared anesthetic cocktail (55% ketamine, 28% xylazine, 11% acepromazine). L. monocytogenes was injected directly into the lateral ventricle of the brain using a KOPF stereotaxic frame (Tujunga, CA) with coordinates: þ1.5-mm lateral from bregma and 4.7-mm below the skull surface. Animals were allowed free access to food and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
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water from this time until killed at 24, 48, or 72 h after bacterial administration.
L. Monocytogenes Inoculum Preparation and Bacterial Burden Determination For each experiment, a Listeria aliquot was thawed and plated onto a 5% Trypsin Soy Agar blood plate, followed by an overnight incubation at 378C. Isolated bacterial colonies were then selected, inoculated into 4 mL of sterile BHI, and incubated at 378C with vigorous shaking for 12 h. After this incubation period, 50 mL were placed into 5 mL of fresh BHI broth and incubated for 3 h at 378C with shaking, to reach the exponential phase of bacterial growth. Bacterial growth and density of the solution were then determined by an absorbance determination of a 1:10 dilution of the broth using a wavelength of 620 nm. An absorbance of 0.6 corresponds to a solution containing 5 108 colony forming units (CFU) of bacteria/milliliter.17 Rats were injected icv with 5 102 CFU of L. monocytogenes in a total volume of 10 mL and control animals received an equal volume of 0.9% sterile saline. Bacterial counts in tissues were used to define the extent of infection at the time rats were killed. Liver and brain samples were weighed and homogenized in 5 mL of sterile phosphate buffered saline under sterile conditions. Dilutions of the homogenates were then made in sterile 0.9% NaCl and plated onto blood agar plates. The plates were incubated at 378C for 24 h and the number of colonies grown was counted at specific dilutions. Bacterial count was expressed as CFU/milligram of tissue.
Tissue Preparation Hepatic microsomes were prepared by the method described by el-Masry et al.20 Animals were killed 24, 48, or 72 h post-treatment by CO2 asphyxiation; livers were rinsed with cold 1.15% KCl and homogenized in 20 mL of the same solution with a Polytron homogenizer. The homogenates were centrifuged at 10,000g for 10 min at 48C and the supernatants were re-centrifuged at 110,000g for 40 min. The resulting pellets were resuspended in 2.5 mL of glycerol buffer (50 mM monobasic KH2PO4, pH 7.5, 20% glycerol, 0.40% KCl) using a glass homogenizer. The suspension was divided into 750-mL aliquots and stored at 708C. Microsomal protein content was determined by the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
method of Lowry et al.21 using bovine serum albumin as a standard. Cytochrome P450 Activities CYP1A1/2 activity of liver microsomes was determined by the formation of resorufin from ethoxyresorufin and CYP2B1/2B2 activity was determined by measuring the formation of resorufin from benzyloxyresorufin.22 CYP3A2 activity of hepatic microsomes was determined by the formation of 4-OH midazolam (MDZ) from MDZ detected by high-performance liquid chromatography.23–25 Reaction mixtures contained 50 mM phosphate buffer, 1 mM NADPH, 0.5 mg of microsomal protein/milliliter, and 12.5 mM MDZ. Formation of the 4-OH MDZ metabolite was linear for up to 5 min and a microsomal protein concentration of 2 mg/mL. Reactions were stopped with 5 mL of methylene chloride and the internal standard, phenacetin, was added. The samples were mixed, centrifuged, and the organic phase was transferred to glass tubes to be evaporated under a nitrogen stream. Samples were resuspended in 125 mL of mobile phase (29% methanol: 28% acetonitrile: 41% 0.01 M KH2PO4, pH 7.4: 2% tetrahydrofuran). A volume of 50 mL of resuspended product was injected on to a Beckman C8 column (5 mm 4.5 mm 25 mm i.d.) using a UV detector at 220 nm to detect the metabolite. All activities were normalized to the amount of protein in each sample. Hepatic P450 Content Determination Hepatic microsomal total cytochrome P450 content was determined as described by the method of Omura and Sato26 using a molar extinction coefficient for the reduced P450-CO complex of 91 cm1 mM1. Immunohistochemistry Twenty-four and 48 h after icv injection of L. monocytogenes or saline, rats were anesthetized with approximately 70 mg/kg pentobarbital and perfused transcardially with 60 mL of 0.9% saline followed by about 120 mL of cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4). Whole brains were carefully removed and placed into vials containing paraformaldehyde for 24 h, followed by cryoprotection in 30% sucrose at 48C until completely submerged. Brains were then sectioned into 40-mm coronal sections with a
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freezing microtome and stored at 48C in Millonig’s solution until further processing. Heat shock protein 27 (hsp27) staining was performed on the sections according to the protocol described by Garcion et al.27 using a rabbit antimouse hsp25 polyclonal antibody (1:5000 dilution in 2% goat serum) (StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) This antibody cross-reacts with the rat hsp27. The secondary antibody was biotinylated goat antirabbit immunoglobulin (Ig)G antibody (1:400 dilution in PB) detected with Avidin Biotin solution, followed by exposure to 3,30 -diaminobenzidine tetrahydrochloride. Western Blot Analysis Each lane was loaded with a 10-mL volume containing 25 mg of microsomal protein. Samples were separated on a 7.5% Tris-HCl Ready Gel (Bio-Rad Labs, Hercules, CA) under nonreducing conditions. The proteins were then transferred to an Immobilon P membrane using a wet transfer. CYP3A1/2 was detected using a mouse anti-rat CYP3A1/2 monoclonal antibody. Membranes were blocked for 2 h at room temperature (RT) in 5% skim milk, followed by incubation with the primary antibody (1:5000 dilution) in 2% skim milk for 1 h at RT. A peroxidase-conjugated anti-mouse IgG secondary antibody was used to proceed with the immunological detection of the protein using standard procedures. CYP1A1/2 and CYP2B1/2 were detected using goat anti-rat CYP1A1/2 and CYP2B1/2 polyclonal antibodies, respectively. Membranes were blocked for 2 h at RT in 5% skim milk containing 0.5% bovine serum albumin, followed by incubation with the primary antibody (1:500 dilution) in 2.5% skim milk for 1 h at RT. The secondary antibody used was an anti-goat IgG conjugated to peroxidase. Bands were visualized using Supersignal Ultra chemiluminescent substrate (Pierce, Rockford, IL). Blots were exposed to X-ray film and band intensities measured using the program Molecular AnalystTM (Bio-Rad, Mississauga, Ontario, Canada). A set of Kaleidoscope prestained molecular weight standards (Bio-Rad, Mississauga, Ontario, Canada) was used for all blots to determine the size of the bands detected. Statistical Analysis Comparisons between groups were performed using two-way analysis of variance, followed by
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Bonferroni post-tests16 in all figures. A p value < 0.05 was considered significant. Error bars on all graphs represent the standard error of the mean of that group.
RESULTS L. Monocytogenes Infection and Bacterial Load in Brain and Liver A dose of 5 102 CFU of L. monocytogenes injected into the lateral ventricle of the brain produced a nonlethal infection, and no severe morbidity was observed throughout the duration of these experiments. The rats injected with L. monocytogenes, however, had signs of lethargy, decreased social interaction, decreased grooming, and a slight weight loss. The time course of the infection was monitored by measuring the number of bacteria present in the brain and liver during the first 72 h after administration of the organism. The number of CFU of bacteria found per milligram of tissue in the brain increased rapidly from 3.6 103 CFU at 24 h, 3.7 104 CFU at 48 h, and reached over 4 104 CFU by 72 h. Only a small number of bacteria were detected per milligram of liver at 24 h (4 10 CFU) and 48 h (1.7 102 CFU) after Listeria infection and this increased to 1.3 103 CFU at 72 h. No bacteria were detected in control animals that received saline. Expression of hsp27 in the Brain after L. Monocytogenes Infection The development of an inflammatory response to infection in the brain was performed by assessing immunohistochemical staining of coronal brain sections with an antibody against hsp27. Immunoreactivity to hsp27 was expressed in the areas surrounding the ventricles and in the parenchyma next to these regions at 24 and 48 h after infection (Figs. 1 and 2, respectively). Immunoreactivity in the corresponding sections taken from control animals treated with saline was low. Effect of CNS Listeriosis on CYP1A, CYP2B, and CYP3A At 48 h after infection, the ethoxyresorufin deakylase activity (CYP1A) was increased by 80% but remained unchanged at 24 and 72 h (Fig. 3a). Benzyloxyrexorufin de-alkalase (CYP2B) and MDZ hydroxylase activity (CYP3A) remained JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
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Figure 1. Expression of hsp27 around the lateral (A and B) and third (C and D) ventricles 24 h after icv administration of 5 102 CFU of L. monocytogenes. Panels A and C correspond to brain sections from control animals, whereas panels B and D are the counterparts from L. monocytogenes infected animals. All images were taken at an original magnification of 5.
unchanged for 24 and 48 h after infection before decreasing at 72 h (Fig. 3b,c). No changes in CYP1A1/2 or CYP2B1/2 protein content were observed at any of the time points studied as reflected by Western blot analysis (Fig. 4a,b) whereas CYP3A1/2 content was significantly decreased at 72 h after L. monocytogenes administration (Fig. 4c). A decrease in total P450 levels in the liver was observed after a 48-h infection period (Fig. 5).
DISCUSSION It has now been established that systemic inflammation and infections can trigger a mechanism resulting in a direct or indirect alteration in cytochrome P450 forms and their corresponding activities.1,5 This results in changes in the capacity of the liver to metabolize drugs and chemicals and has a profound effect on the pharmacokinetics of certain drugs. Hepatic P450 regulation also seems to be compromised during inflammaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
tory responses originating in the brain.5–7 The mechanism responsible for the signaling between the CNS and the periphery is complicated and may involve the expression of cytokines, prostaglandins, and other mediators.5 The current study suggests that changes to drug biotransformation in the liver also occur during bacterial infections that are largely confined to the CNS. Infection of the brain by L. monocytogenes produced changes in a number of cytochrome P450 forms of the enzyme in the liver. The infection was confirmed by the recovery of bacteria from the brain stem of the infected rats 24, 48, and 72 h after icv administration, and a high immunoreactivity against hsp27 in the brains of infected animals. In contrast, the number of bacteria found in the liver at these times was small, indicating that a generalized systemic infection did not occur. In terms of the activities of CYP450 in the liver, different effects were observed at different points after icv injection, which may be an indication of the stage of development of infection. CYP1A activity shows a strong though temporary increase, 48 h after
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Figure 2. Expression of hsp27 around the lateral (A and B) and third (C and D) ventricles 48 h after icv administration of 5 102 CFU of L. monocytogenes. Panels A and C correspond to brain sections from control animals, whereas B and D are the counterparts from L. monocytogenes infected animals. All images were taken at an original magnification of 5.
infection, which returned to control levels by 72 h. This pattern is similar to that described for a L. monocytogenes systemic infection in mice by Armstrong and Renton,19 who showed a significant increase in CYP1A mRNA and protein at 12 and 24 h, followed by a profound loss in enzyme during the period when the bacterial level in the liver was very high. The delayed onset in our study (i.e., 48 vs. 12 h) is likely attributed to the time required for the infection to develop and produce signaling molecules responsible for the hepatic loss. Both CYP2B and CYP3A activities were significantly reduced 72 h after Listeria administration, but only CYP3A microsomal protein was altered. Although the bacterial load in the liver was relatively small at this time, it had increased to more than 1 103 CFU/mg liver and it is possible that the response to the increasing number of organisms was responsible for the loss in CYP3A at this time. The apparent discrepancies between the protein levels for specific cytochrome P450 forms and the corresponding enzyme activities observed at certain times in these experiments is difficult to
explain but similar discrepancies for several forms of cytochrome P450 have previously been reported in a number of studies using LPS and a Staphylococcal enterotoxin.28–30
CONCLUSIONS These experiments show for the first time that a live bacterial infection largely confined to the CNS modulates cytochrome P450 enzymes in the liver of the rat. The loss of enzyme in the liver is time and bacterial load dependent, which reflects the dynamic nature not only of disease, but also of the concomitant regulation of cytochrome P450 enzymes that follows. The observation that total hepatic cytochrome P450 content was significantly lowered 48 h after infection implies that other isoforms, besides those we analyzed, may be affected by this condition. If this occurs in humans, the implications of these findings to the medical community are important. The number of drugs known to be metabolized by these JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
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Figure 3. Effect of CNS infection by L. monocytogenes on hepatic CYP1A (a), CYP2B (b), and CYP3A (c) activities. Male Sprague-Dawley rats received a single icv injection of 5 102 CFU of L. monocytogenes contained in a total volume of 10 mL of sterile 0.9% NaCl. Control animals received 10 mL of 0.9% NaCl. Rats were killed 24, 48, and 72 h after infection. Values are reported as percent of control SEM (n ¼ 8 for 24 h, nsaline ¼ 8, nlisteria ¼ 9 for 48 h, and n ¼ 4 for 72 h). ***Significantly different from control levels, p < 0.001; **Significantly different from control levels, p < 0.01; *Significantly different from control levels, p < 0.05. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 9, SEPTEMBER 2003
Figure 4. Effect of CNS infection by L. monocytogenes on hepatic protein content of CYP1A (a), CYP2B (b), and CYP3A (c). Treatment and groups were identical to that described in Figure 3. **Significantly different from control values, p < 0.01.
enzymes is numerous and may lead to an unpredictability of drug metabolism in individuals with a CNS bacterial infection. Because a significant proportion of the population is at risk for CNS listeriosis, the number of potential drug
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7.
8.
9. Figure 5. Effect of CNS infection by L. monocytogenes on total hepatic P450 content. Treatment and groups were identical to that described in Figure 3. *Significantly different from control levels, p < 0.05.
toxicities in these individuals could be a serious problem in therapeutics.
ACKNOWLEDGMENTS The authors thank Rafael and Elizabeth Gardun˜o for kindly providing the L. monocytogenes strain, as well as their expertise and patience. E. Garcia del Busto Cano was a recipient of a Killam Trusts studentship. This work was supported by the Canadian Institute of Health Research (CIHR). A portion of this work was presented in abstract form at the ASBMB/ASPET/FPS/PSC 2000 joint meeting in Boston, MA.
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