Depression of Hepatic Mixed-Function Oxidase Activity by B. pertussis in Splenectomized and Athymic Nude Mice

Joseph F. Williams, Alvin L. Winters, Saul Lowitt, and Andor Szentivanyi

Abstract: Administration of Bordetella pertussis (B. pertussis), Corynebacterium parvum (C. parvum), and several other immunoa~ve substances is known to cause a marked decrease in the activity of the hepatic microsomal mixed-function oxidase system. The effect of C. parvum has been reported to involve the reticuloendothelial system. In the present study, the effect of B. pertussis administration to decrease hepatic microsomal drag metabolism was studied in unoperated, sham-operated, and splenectomized mice as well as in athymic nude (nu/nu) mice and their phenotypically heterozygous ( +/nu) littermates. Administration of B. pertussis to the splenec- tomized, sham-operated, and unoperated mice resulted in a decrease in the activity of the microsomal enzyme system that was approximately the same for each of the three groups of animals. Administration of B. pertussis to nu/nu mice and the +/nu mice also decreased the microsomal enzyme activity measured 24 hr after injectionl However, at 7 days after B. pertussis administration, the hepatic drug-metabolizing activity of the nu/nu mice was not significantly different from control values, whereas the activity of the +/nu mice was still significantly depressed. The failure of splenectomy to prevent the decrease in microsomal mixed-function oxidase activity caused by B. pertussis indicates that the effect of this agent differs from that of C. parvum, whose effect was prevented by splenectomy. Indeed, the results obtained with the athymic nude mouse suggests that the depression of hepatic mixed-function oxidase activity by B. pertussis may involve T-cell dependent responses.

Key Words: B. pertussis; Hepatic mixed-function oxidase activity; Athymic mice; Splenectomy

INTRODUCTION

The biotransformation of many drugs, exogenous chemicals, and certain endogenous com- pounds can be increased or decreased by factors that affect the cytochrome P-450-dependent mixed-function oxidases in the endoplasmic reticulum of the liver (Gillette et al., 1972). It is well-known (Gillette et al., 1972) that exposure of animals or man to a wide variety of drugs or

Received April 7, 1980; revised August 15, 1980. From the Depar~nent of Pharmacology and Therapeutics and the Department of Medical Microbiology,

University of South Florida College of Medicine, Tampa, Florida. Address requests for prints to: Dr. Joseph F. Williams, Department of Pharmacology & Therapeutics,

University of South Florida College of Medicine, 12901 N. 30th Street, Tampa, FL 33612.

© Elsevier North Holland, Inc., 1981 101 52 Vanderbllt Ave., New York, N.Y. immunopharmacolo~ 3, 101-106 (1981) 0162-3109/81/02010106502.50

102 J .F. Williams et al.

other xenobiotics can induce the hepatic drug-metabolizing enzyme activity. Decreased hepatic drug-oxidation reactions have been reported following treatment with nonspecific im- munomodulants, such as Freund's complete adjuvant (Beck and Whitehouse, 1973; Cawth- rone et al., 1976), Bacillus Calmette-Guerin (Farquhar et al., 1976), endotoxin (Vanio, 1973; Gorodischer et al., 1976; Renton and Mannering, 1976; Williams et al., 1980), poly rl-poly rC (Renton and Mannering, 1976), Corynebacterium parvum (Soyka et al., 1976), and Bordetella pertussis (Williams and Szentivanyi, 1975; Renton and Mannering, 1976; Williams et al., 1977; Williams and Szentivanyi, 1977; Williams et al., 1980).

The mechanism by which the various immunomodulators cause the decreased hepatic mixed-function oxidase activity is unknown. One consideration is that the effect of these agents on this hepatic enzyme system may be related to their effects on cellular constituents in the immune system, as previously suggested by Farquhar et al. (1976) and Mathur et al. (1978). Recently Soyka et al. (1979) reported that the effect of C. parvum on hepatic drug-metabolizing activity may be related to an effect on this agent on macrophages. Procedures that are thought to,alter macrophage function, e.g., prior splenectomy, injection of silica, and whole body irradiation, were shown to markedly diminish the ability of C. parvum to alter the activity of the drug-metabolizing system. Thus, Soyka and co-workers suggested that cells of the reticuloen- dothelial system (RES) may play a role in the depression of the hepatic mixed-function oxidase system produced by C. parvum.

The present article has examined the possible involvement of two organs of the immune system in the decreased hepatic drug-metabolizing activity produced by B. pertussis vaccine. First, as suggested from the report of Soyka et al. (1979), the effect of pertussis vaccine on hepatic mixed-function oxidase activity was examined in splenectomized mice. Secondly, the possible involvement of the thymus in the effect of pertussis was studied using congenitally athymic (nude) mice and their heterozygotic littermates.

MATERIALS AND METHODS

Female CD-1 mice, 18-20 g, (Carworth Farms, Portage, MI) were maintained under standard laboratory conditions. Splenectomy was performed under ether anesthesia 48 hr prior to injection of B. pertussis (Soyka et al., 1979). Sham-operated mice served as controls.

A breeding colony of athymic mice of the C3H/HeN strain was obtained from Dr. Carl Hansen (National Cancer Institute, Bethesda, MD) and maintained in our facilities according to a modification of the procedure of Giovanella and Stehlin (1973). The animals were housed at 25-30°C in individual autoclaved polycarbonate cages fitted with a filter cover. Autoclaved mouse chow and water was supplied ad libitum. Food and water addition to the cages and all manipulation of the animals were performed using aseptic procedures. Animals used in the experiments were 6- to 12-week-old female, athymic, nude mice (nu/nu) and their phenotypi- cally heterozygous (+/nu) littermates.

B. pertussis vaccine (Eli Lilly & Co., Indianapolis, IN) was diluted 1:1 with sterile, pyrogen-free isotonic saline. Groups of animals received intraperitoneal (ip) injection (0.35 ml) of the diluted vaccine (7 x 109 cells/mouse); controls received the diluent only. Animals were killed by cervical dislocation, livers were excised and gall bladders removed. Livers from three animals per group were pooled, minced with scissors, and homogenized in 4 volumes of cold 1.15% KCI using a Dounce homogenizer fitted with a glass pestle (10 strokes). The homogenate was centrifuged for 20 rain at 9000xg and the resulting supernatant was centrifuged for 60 rain at 105,000xg. The microsomal pellet was resuspended in 1.15% KCI solution. Microsomal

Abbreviations. B. pertussis: Bordetella pertussis; C. paruum: Corynebacterium paruum; nu/nu: athymic nude mice; +/nu: heterozygous animals; RES: reticuloendothelial system.

B. pertussis and Decreased Drug-metabolizing Activity 103

protein was determined by the method of Lowry et al. (1951) using bovine serum albumin (Sigma Chemical Co., SL Louis, MO) as reference standard.

Ethylmorphine N-demethylase and aniline hydroxylase activity were determined as previ- ously reported (Williams et al., 1980). Cytochrome P-450 concentration was measured according to the procedure of Omura and Sato (1964)using an extinction coefficient of 91 cm-lmM -1 for the difference between 450 and 490 nm.

Statistical comparison was performed using Student's t-test for independent samples.

RESULTS

The N-demethylation of ethylmorphine, the hydroxylation of aniline, and the level of cytochrome P-450 was examined in unoperated, sham-operated, and splenectomized mice 7 clays after pertussis administration (Table 1). Sham operation and splenectomy were performed 48 hr before pertussis vaccine administration. In agreement with the observations of Soyka et al. (1979), neither sham operation nor splenectomy per se caused a significant change in the drug-metabolizing enzyme activity or in the level of cytochrome P-450. Seven days after pertussis administration all three groups of animals showed a decrease in the rate of ethylmorphine N-demethylase activity, aniline hydroxylase activity, and the level of cytochrome P-450 that was significantly different from that observed in the nonvaccinated groups. In addition, no difference was noted in the spleen or liver weights of the pertussis-treated mice from that observed in control animals. There was also no difference between the control and experimental groups in the level of microsomal protein. Comparable results to those shown in Table 1 were also obtained if the vaccine was administered intravenously (data not shown).

Table 2 gives the results of studies of the hepatic mixed-function oxidase activity in the athymic nude (nu/nu) mouse and their heterozygous (+/nu) littermates. No significant difference in enzyme activity or cytochrome P-450 levels was noted between nu/nu and +/nu control animals. A significant decrease in the rate of ethylmorphine N-demethylation and in the level of cytochrome P-450 was observed at 24 hr after pertussis administration to both the nu/nu and the +/nu groups of mice. The rate of aniline hydroxylation was not statistically affected by the pertussis administration. Seven days after the injection of B. pertussis to the

Table 1 Effect of B. pertussis administration to splenectomized mice on hepatic drug-metabolizing enzyme system a

Animals Pertussis Ethylmorphine Aniline Cytochrome injected N-demethylase hydroxylase P-450

activity b activity c (nmol/mg protein)

Unoperated control - 0.369_+0.025 44 .01+3 .23 0.80-+0.07 + 0 .189±0 . 019 a 35.79__-1.42 • 0 .68±0 .04 a

Sham-operated - 0.331 ±0 .007 38 .25± 1.63 0 .82±0 .08 + 0.206_+0.028 d 27.66+_3.27 e 0.46-+0.01 a

Splenectomized - 0 .344±0 .018 40 .25±3 .48 0 .78±0 .03 + 0 .259±0 .010 d 26.78-+1.38 e 0 .53±0.01 d

a Animals were injected ip with 7 x 109 organisms/mouse 48 hr after surgery and sacrificed 7 days after injection. Tabular values are mean - SE of 4 experiments with 3 animals per group.

b Micromoles formaldehyde formed/mg microsomal protein/hr.

c Nanomoles paraaminophenol formed/mg microsomal protein/hr.

alp<0.01.

ep<O.OS.

104 J .F . Williams et al.

Table 2 Effect of B. pertussis administration to athyrnic mice (nu/nu) and animals heterozygous for the athymic trait ( + /nu) on the hepatic drug-metabolizing enzyme system °

Ethylmorphine Aniline Cytochrome N-demethylase hydroxylase P-450

activity b activity c (nmol/mg)

Heterozygous (+/nu) Control

i day 0.214-+.017 41.15-+2.93 0.84+_0.05 7 day 0.197-+0.018 35.96-+1.51 0.80-+0.06

Pertussis 1 day 0.125_+0.009 d 36.74_+2.59 0.56_+0.06 d 7 day O. i05--0.010 a 34.08-+1.53 0.48-+0.04 d

Athymic (nu/nu) Control

1 day 0.269-+0.030 42.79_+4.32 0.80_+0.08 7 day 0.266-+0.025 43.86-+3.16 0.81-+0.05

Pertussis I clay 0.153+0.009d 36.07-+2.50 0.54-_0.05 e 7 day 0.236-+0.016 39.85-+2.96 0.69-+0.04

a Pertussis vaccine (7 x I09 cells/mouse) was administered ip. Tabular values represent mean _+ SE of 6 - 8 experiments with 3 animals per group.

b Micromoles formaldehyde formed/rag/flicrosomal protein/hr.

c Nanomoles paraaminophenol formed/rag microsomal protein/hr.

ap<O.01.

~p<O.05.

+/nu animals, the activity of the microsomal drug-metabolizing enzyme system and the level of cytochrome P-450 were still significantly depressed. In ,contrast, the rate of ethylmorphine N-dem~thylation and the level of cytochrome P-450 of the nu/nu mice injected 7 days before with the pertussis vaccine was not significantly different from the nu/nu control values.

DISCUSSION

The results of the present study indicate that the inhibitory effect of B. pertussis administration on the hepatic drug-metabolizing system may be elicited by different mechanisms of the immune system than those suggested for C. parvum (Soyka et al., 1979). Prior splenectomy did not alter the effect of B. pertussis administration to decrease hepatic microsomal ethylmorphine N-demethylase or anailine hydroxylase activity or to decrease the level of cytochrome P-450. This effect ofB. pertussis on splenectomized animals is contrasted to the lack of a corresponding effect of C. p, arvum in splenectomized animals. It should be noted that C. parvum administration to mice was also shown to produce hepatosplenomegaly, whereasB, pertussis injection did not.

Athymic nude mice, genetically deficient in T-lymphocytefunction, (Sprent and Miller, 1972; Rygaard, 1973), have been used extensively to investigate T-cell dependency of immunological responses. The transient decrease in hepatic mixed-function oxidase activity produced by B. pertussis in the nu/nu mouse as compared to the persistent decrease in enzyme activity seen in the +/nu mice may be indicative that normal thymus function, e.g., T-lymphocytes, may be

B. pertussis and Decreased Drug-metabolizing Activity 105

required for the full expression of the vaccine effect. Administration of pertussis to nu/nu animals caused a decrease in ethylmorphine N-demethylase activity and a decrease in the level of cytochrome P-450 at 24 hr, but the enzyme activity had returned to control values by 7 days. B. pertussis administration to the +/nu animals produced a comparable decrease at 24 hr and in these animals the decreased enzyme activity was also seen at 7 days after injection.

The transitory depression of the hepatic drug-metabolizing activity observed in the present study with the nu/nu mice is similar to that previously reported (Williams et al., 1980) using conventional mice injected with B. pertussis vaccine that had been heated to 80°C for 0.5 hr. It appears that two distinct components, one heat-stable and the second heat-labile, are involved in the effect of B. pertussis to decrease hepatic drug-metabolizing activity. The heat-stable component, possibly the endotoxin component of this gram-negative organism, appears to be responsible for the early decrease in enzyme activity seen 24 -36 hr after injection. The identity of the second, 80°C-heat-labile component involved in causing the prolonged loss of drug-metabolizing activity is unknown. However, it is evidently not the histamine-sensitizing factor (pertussigen), known to be similarly heat-labile, since injection of partially purified pertussigen did not alter enzymatic activity (Williams et al., 1980). The results of the present study would suggest that both the nu/nu and the +/nu mice are responsive to the heat-stable (endotoxin) component, but that the nu/nu mice are unresponsive to the heat-labile compo- nent. It is of interest that a heat-labile component of pertussis has been shown to be necessary for sustained modulation of acid phosphatase activity in adherent mononuclear cells (Dr. T. Klein, personal communication). Furthermore, nu/nu mice injected with pertussis showed an early modulation (2 days) of acid phosphatase activity, but not a sustained response, These observations are in line with the demonstrated requirement for T lymphocytes for microbial products to modify macrophage function (David and Remold, 1976).

Although the results strongly suggest that the T cells may be involved in the response to B. pertussis, other considerations relative to the use of the nu/nu mouse must also be assessed. For example, nu/nu mice have been shown to possess activated macrophages (Meltzer, 1976). An increased phagocytic clearance of injected pertussis organisms could conceivably reduce the time for the effect on hepatic activity and, thus, result in a transient decrease in enzymatic activity. Certainly, further work will be necessary to examine this possibility.

The apparent association of immunomodulation and regulation of hepatic drug-metabolizing activity by B. pertussis and other immunoactive agents warrants further study. Such studies may provide additional information concerning regulatory mechanisms of the hepatic mixed-function oxidase system not previously recognized. Additionally, an effect of immunoactive agents to depress drug-oxidation reactions may have clinical relevance during the combined immuno- and chemotherapeutic treatment of cancer, where drug-metabolizing activity can be important for either the activation or inactivation of therapeutic agents.

The authors wish to thank Ms. Bettye Bing and Mr. Jerry Perry for their technical assistance. This work was supported in part by Biomedical Research Support Grant NIH 5 SO7 RR05749-05.

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