Joseph F. Williams

Differential effect of cytokines on the phenobarbital or 3-methylcholanthrene induction of P450 mediated monooxygenase activity in cultured rat hepatocytes

Cultured rat hepatocytes have been used to compare the relative activities of cytokines to inhibit the phenobarbital (PB) or 3-methylcholanthrene (MC) induction of cytochrome P4502B1 and 2B2 (P4502B1/2) or P4501A1 and 1A2 (P4501A1/2), respectively. Recombinant cytokines tested were human interleukin-6 (IL-6), interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta, respectively), and rat gamma-interferon (INF gamma). Hepatocytes were cultured in the presence of 2 mM PB or 1 microgram MC/mL culture medium for 24 hr with or without the cytokines. Benzyloxyresorufin and ethoxyresorufin O-dealkylase (BROD and EROD, respectively) activities were determined as indices of P4502B1/2 and P4501A1/2, respectively. All cytokines produced a concentration-dependent inhibition of the PB induction of BROD activity. IL-1 beta and IL-6 were approximately equipotent with IC50 values of 1-2 U/mL, causing greater than 90% inhibition of PB induction of BROD activity at a concentration of 50 U/mL culture medium. IL-1 alpha tended to be less active. PB induction of BROD activity was also inhibited by INF gamma, but higher concentrations (62.5 to 500 U/mL culture medium) were required. All cytokines were less effective in inhibiting the MC induction of EROD activity than the PB induction of BROD activity. IL-1 beta and IL-6, at 50 U/mL culture medium, inhibited EROD induction by only 35% compared with the greater than 90% inhibitory effect on the PB induction of BROD activity. INF gamma was ineffective in inhibiting EROD activity at the concentrations studied. Western immunoblot analysis indicated that the cytokines prevented the ability of the inducers to increase the expression of P4502B1/2 and P4501A1/2 immunoreactive proteins, and this effect correlated with their inhibitory effect on induction of enzyme activity. The results suggest that inducible isoforms of cytochrome P450 differ in their susceptibility to regulation by the cytokines, and that cytokines possess differential activity to inhibit the induction of P450 isoforms, with IL-1 beta and IL-6 being the most effective.

The Chronic Administration of Nicotine Induces Cytochrome P450 in Rat Brain

Abstract: The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague‐Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c, twice daily for 10 days), and total cytochrome P450 content, the activity of N ADPH‐cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine‐injected rats: the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH‐cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O‐dealkylase (BROD) and pentoxyresorufin O‐dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213‐227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH‐cytochrome c reductase, BROD, and PROD, and levels of P4502B1 /2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.

Effects of phenobarbital and 3-methylcholanthrene pretreatment on the plasma half-life and urinary excretion profile of theophylline and its metabolites in rats

Abstract The effects of the inducers of the hepatic microsomal enzyme system, phenobarbital and 3-methylcholanthrene, on theophylline plasma half-life and on the elimination of theophylline and its metabolites in urine and feces have been examined. The results indicate that induction of the hepatic microsomal drug-metabolizing enzyme system significantly decreases plasma theophylline half-life. In this respect, 3-methylcholanthrene was more effective than phenobarbital. Control theophylline half-life was 3.5 hr. After phenobarbital or 3-methylcholanthrene pretreatment, the theophylline half-life was 2.6 and 0.8 hr respectively. Thin-layer Chromatographie analysis of the urine showed three radioactive peaks corresponding to 1,3-dimethyluric acid, 1-methyluric acid and unchanged theophylline. Both inducing agents significantly increased the urinary elimination of 1,3-dimethyluric acid above that seen in control animals throughout the 24-hr collection period, but only 3-methylcholanthrene increased the total amounts of 1-methyluric acid excreted. Urinary elimination of unchanged theophylline was decreased from control values by both agents. A small, but not statistically significant, increase in the fecal elimination of radioactive material was also noted in the animals pretreated with phenobarbital. The results indicate that alteration in hepatic drug-metabolizing activity may markedly affect the in vivo biotransformation of theophylline.

Effect of interleukin 6 on phenobarbital induction of cytochrome P-450IIB in cultured rat hepatocytes.

Human recombinant interleukin 6 (rhIL-6) caused a dose dependent decrease in the phenobarbital induction of benzyloxyresorufin O-deethylase activity in cultured rat hepatocytes. Decreased enzymatic activity was associated with a decrease in the amount of immunoreactive P-450IIB1/2. rhIL-6 also prevented the PB-induced increase in the steady state level of P-450IIB mRNA. These results suggest that altered P-450 levels observed in vivo during the acute phase reaction may be due to interleukin 6.

Pharmacological and biochemical activities of some monomethylxanthine and methyluric acid derivatives of theophylline and caffeine

Abstract Some monomethylxanthine and methyluric acid derivatives of theophylline and caffeine have been studied to explore whether they possess pharmacological and biochemical activities similar to those of their parent compounds. Both 3-methylxanthine and 1-methylxanthine, but not 1,3-dimethyluric acid or 3-methyluric acid, produced the same maximal relaxation of guinea pig tracheal muscle as did theophylline. The EC 50 values for theophylline and 3-methylxanthine were not significantly different, whereas those for 1-methylxanthine, 1,3-dimethyluric acid and 3-methyluric acid were significantly higher than that if theophylline. In the Langendorff guinea pig heart, theophylline and 3-methylxanthine caused essentially identical increases in cardiac contractile force. Although less effective than theophylline, 1-methylxanthine and caffeine produced equivalent increases in cardiac contractility. At concentrations higher than those effective for the methylxanthines, 1,3-dimethyluric acid markedly increased contractile force. 3-Methylxanthine inhibited cyclic AMP phosphodiesterase to a lesser extent than did theophylline at both 1.4 and 400 μM cyclic nucleotide concentrations. However, at the higher substrate concentration, cyclic GMP phosphodiesterase activity was inhibited by 3-methylxanthine more than by theophylline. Thus, it appears that the monomethylxanthine and methyluric acid derivatives of theophylline and caffeine possess a spectrum of pharmacological activity similar to that of their parent compounds, a finding which raises important questions about various aspects of the current therapeutic use of methylxanthines.

Induction of tolerance in mice and rats to the effect of endotoxin to decrease the hepatic microsomal mixed-function oxidase system. Evidence for a possible macrophage-derived factor in the endotoxin effect

Daily administration of low, non-lethal doses of bacterial endotoxin to mice and rats has been shown to induce tolerance to the effect of an acute challenge dose of endotoxin to decrease the hepatic microsomal drug metabolizing activity, the level of cytochrome P-450, and to increase heme oxygenase activity. The serum collected at various times after injection of endotoxin into control animals when injected into untreated animals markedly depressed aniline hydroxylase activity, ethylmorphine N-demethylase activity, and the level of cytochrome P-450. Tolerant animals were not affected by the post-endotoxin serum injection, suggesting the decreased activity caused by the serum in untreated animals was probably due to endotoxin contained in the serum. Injection of tolerant mice and rats with supernatant medium obtained from cultures of peritoneal macrophages incubated with 100 micrograms/ml of endotoxin caused a loss of hepatic microsomal drug-metabolizing activity, and a decrease in the level of cytochrome P-450. These results suggest that peritoneal macrophages release a factor in response to endotoxin that mediates the decreased hepatic mixed-function oxidase activity.

Involvement of a heat-stable and heat-labile component of Bordetella pertussis in the depression of the murine hepatic mixed-function oxidase system

Abstract Experiments were conducted to determine whether the decrease in ethylmorphine N -demethylase and aniline hydroxylase activities and in the levels of cytochrome P-450 observed after injection of Bordetella pertussis to mice was related to an activity of the well-characterized 80°-heatlabile bacterial component (HSF) which causes an increased sensitivity to histamine. Temporal studies over 10–15 days following B. pertussis inoculation of mice suggested a possible correlation between the development of histamine sensitivity and the decrease in both in vivo and in vitro activities of the hepatic mixed-function oxidase system. Treatment of mice with unheated or 56°-heated vaccine produced a decrease in microsomal drug-metabolizing enzyme activity and an increased sensitivity to histamine at both 24 hr and 5 days after injection. In contrast, mice injected with an 80°-heated vaccine did not show an increased sensitivity to histamine at either time point or a decrease in drug-metabolizing activity at 5 days. There was, however, a significant loss of microsomal enzyme activity determined at 24 hr post-injection of the 80°-heated vaccine. Injection of B. pertussis into a strain of mice insensitive to HSF activity was shown to produce a decrease in the drug-metabolizing activity at both 24 hr and 5 days without the concomitant increase in histamine sensitivity. On the other hand, injection of partially purified HSF into a susceptible strain of mice produced histamine sensitization without the loss of hepatic enzyme activity. These studies suggest that HSF per se is not the bacterial component responsible for the reduction in the hepatic microsomal enzyme activity. The results would indicate that two separate bacterial components may be involved. One is stable to heat of 80° and may cause the acute (24 hr) loss of activity. The second is labile to heat between 56 and 80° and may be responsible for the prolonged (5–10 days) loss of enzyme activity. The identity of the heat-labile component is unknown but the former, heat-stable component may be bacterial endotoxin. This conclusion is based on the similarity between the transient reduction in drug-metabolizing activity produced by injection of 500 μmg/kg of endotoxin and that produced by the 80°-heated vaccine.

Endotoxin depression of hepatic mixed function oxidase system in c3h/hej and c3h/hen mice.

The effect of endotoxin to depress the hepatic drug-metabolizing enzyme activity has been studied in the C3H/HeJ and C3H/HeN strains of mice. The C3H/HeJ mouse strain is generally considered to be unresponsive to the biological effects of endotoxin. However, injection of these mice with 0.5 mg/kg body weight of E.coli endotoxin (Westphal extracted) produced a decrease in the rate of N-demethylation of ethylmorphine and in the levels of cytochrome P-450 and cytochrome b5 comparable to that observed in the endotoxin-sensitive C3H/HeJ mouse strain. Although the mechanism of endotoxin action to decrease hepatic microsomal drug-metabolizing activity is presently unknown, the results suggest that: 1) the C3H/HeJ mouse stain is responsive to this endotoxin effect, and 2)that cellular constituents other than B-cells or macrophages are probably involved in eliciting the response, since these cells of the C3H/HeJ mouse are unresponsive to endotoxin.

Depression of hepatic drug-metabolizing enzyme activity by B. pertussis vaccination

In vitro measurements are described on N-demethylation of aminopyrine and ethylmorphine, the hydroxylation of aniline as well as the level and activity of the electron transport system in hepatic microsomal fractions from pertussis vaccinated mice. Results indicate that pertussis vaccination of mice markedly decreases the level and activity of the hepatic drug-metabolizing system.

Effects of phenobarbital and interleukin-6 on cytochrome P4502B1 and 2B2 in cultured rat hepatocytes

The objectives of this study were to characterize further the effects of phenobarbital (PB) on cytochrome P4502B1 and 2B2 (P4502B1/2) enzyme activity and immunoreactivity in rat hepatocytes and to investigate the mechanism(s) mediating the ability of interleukin-6 (IL-6) to inhibit this induction. PB caused a concentration-dependent increase in benzyloxyresorufin O-deethylase (BROD) activity with maximal effects (a 25-fold increase) at concentrations of 0.3 to 1 mM. The induction of BROD activity was linear over 24 hr of exposure. Immunoblot profiles of P4502B1/2 agreed with measurements of enzyme activity. In addition to inducing P4502B1/2, PB (0.75 mM) also increased the levels of P450 reductase by approximately 2-fold following a 24-hr exposure to PB. When IL-6 was added concomitantly with or up to 12 hr after the addition of PB, the PB induction of BROD activity and immunoreactivity was inhibited significantly. When 18 hr elapsed between the time of addition of PB and IL-6, the inhibitory effects of IL-6 were no longer apparent, suggesting that the actions of IL-6 were mediated by early events in the induction process. IL-6 did not affect the PB induction of P450 reductase. To determine whether IL-6 altered the degradation of P4502B1/2, hepatocytes were exposed to PB for 24 hr, then washed, and the loss of BROD activity and immunoreactivity following incubation with a protein synthesis inhibitor was measured. IL-6 did not alter the rate of loss of either enzyme activity or immunoreactivity, indicating that the effects of IL-6 could not be attributed to the enhanced degradation of P4502B1/2. Results suggest that the inhibition of PB-induced BROD activity by IL-6 is due to an action on early cellular and molecular events in the induction process.