Yutaka Kikkawa

Effect of interferon inducers on superoxide anion generation from rat liver microsomes detected by lucigenin chemiluminescence.

Microsomal superoxide anion (O2-) production was detected using the chemiluminigenic probe, bis-N-Methylacridinium nitrate (lucigenin). Superoxide dismutase (SOD) inhibited 55% of the light emission but in the presence of a detergent (Triton X100) SOD inhibited the light emission by 94%. Lucigenin chemiluminescence from rat liver microsomes supplemented with NADPH was found to be selective and sensitive in detecting the O2- production. Treatment of rats with poly IC and LPS resulted in a decrease of the hepatic microsomal cytochrome P450 content by 44% and 37% respectively. The decrease in the cytochrome P450 contents was accompanied by a decrease in LgCl from the hepatic microsomal fractions by 61% for the poly IC and by 51% for the LPS treated rats. This is the first report to demonstrate that decreased P450 in the presence of normal amounts of cytochrome P450(c) reductase produce correspondingly less O2- from the microsomes.

Induction of cytochrome P4501A1 by photooxidized tryptophan in Hepa lclc7 cells.

Mouse hepatoma Hepa-lclc7 (Hepa-1) cells were cultivated in the presence of UV-irradiated amino acids. The results demonstrated that all of the amino acids tested, UV-oxidized tryptophan caused the highest induction of 7-ethoxyresorufin O-deethylase (EROD) activity compared with the controls (P < 0.01). The induction of EROD activity by oxidized tryptophan was dose dependent, and maximal induction was obtained at 12 hr after administration. Studies with various Hepa-1 mutants, which are defective in either the aryl hydrocarbon (Ah) receptor or Ah receptor nuclear translocator protein, indicated that the induction of EROD activity by oxidized tryptophan occurs through the Ah receptor. Gel mobility shift assays using nuclear extracts of Hepa-1 cells revealed that oxidized products of tryptophan can induce both Ah receptor transformation and binding of the liganded Ah receptor complex to its specific DNA recognition site. CYP1A1 mRNA, quantified by reverse transcription-polymerase chain reaction, and CYP1A1 protein were induced markedly in the oxidized tryptophan group compared with the controls. Injection of isolated oxidized tryptophan products into adult male rats caused significant induction of EROD activity in the pulmonary and hepatic microsomes compared with the controls (P < 0.01). These results demonstrated that oxidized tryptophan induces Ah receptor activation and binding of the liganded Ah receptor complex to its specific DNA recognition site, thereby initiating transcription and translation of the CYP1A1 gene with concomitant increase of EROD activity in Hepa-1 cells. Induction of EROD activity in the liver and lungs after injection of isolated oxidized tryptophan products into rats suggests that a similar mechanism may be operative in vivo.

Effect of aging on pulmonary superoxide dismutase

Pulmonary Cu,Zn superoxide dismutase was examined in young (1-month-old), adult (4-5-month-old) and aged (24-months-old) rats to determine if partially inactive forms of the enzyme accumulate in the lung with age. Measurement of Cu,Zn superoxide dismutase activity in lung homogenates showed that total Cu,Zn superoxide dismutase activity/mg DNA was essentially the same in adult and aged rats. The average value of Cu,Zn superoxide dismutase/mg DNA for young rats was less than half that of adult and aged rats. Cu,Zn superoxide dismutase was purified from the lung homogenates and fractionated into isoelectric variants by either isoelectric focusing or chromatofocusing. Three main isoelectric variants of Cu,Zn superoxide dismutase were recovered with pI values of 5.15, 4.88 and 4.75. In all age groups studied, the pI 4.88 variant had a markedly higher specific activity than the other two variants, as well as the highest metal content and greatest resistance to inactivation of all three variants. The pI 4.88 variant declined from 88% of the total Cu,Zn superoxide dismutase activity in the young animals to only 70% in the aged animals. The results of this study indicate that the proportion of the relatively inactive forms of pulmonary Cu,Zn superoxide dismutase increased with age.

Hepatic cytochrome P450 enzyme imprinting in adult rat by neonatal benzo[a]pyrene administration

ABSTRACT: The effect of neonatal exposure to benzo[a]pyrene (BaP) on the hepatic cytochrome P450 of male and female adult rats has been examined. Newborn rats (<24 h old) were injected with a single dose of BaP (1 mg/rat, s.c.) and killed after 110 days. In both sexes, body and liver weight, microsomal protein content, and total cytochrome P450 were unchanged. Cytochrome P450 1A2 protein content and 7-ethoxyresorufin O-deethylase activity were significantly decreased (p < 0.05) in males, whereas these were unaltered in females. Male-specific cytochrome P450 2C11 of male rats was significantly increased as shown by Western blot and increased testosterone 2α- and 16α-hydroxylase activities by 29% (p < 0.01) and 22% (p < 0.05), respectively. Female-specific cytochrome P450 2C12 protein content was unaltered in females. In addition, the level of free hepatic glucocorticoid receptor in adult males was elevated by 35% after BaP exposure, whereas it was unchanged in adult females. These results indicate, for the first time, that neonatal BaP exposure results in gender-specific lasting effects on hepatic cytochrome P450 1A2, cytochrome P450 2C11, and glucocorticoid receptors in adult male rats, whereas these parameters are unchanged in adult female rats.,

Role of nitric oxide in the inhibition of cytochrome P450 in the liver of mice infected with Chlamydia trachomatis.

In this study, we attempted to determine the effect of a systemic infection with Chlamydia trachomatis on cytochrome P450(CYP)-dependent metabolism in mice. Furthermore, we wanted to assess if these effects were mediated through NO. BALB/c(H-2d) female mice were inoculated intraperitoneally with the C. trachomatis mouse pneumonitis (MoPn) biovar, and induction of NO synthase (NOS) was detected by measuring [NOx] levels and inducible NOS protein content in peritoneal macrophages by Western blotting. Recovery of C. trachomatis from liver, lung, and spleen peaked at 4 days postinfection. Following cotreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, there was a significant increase in the intensity and the length of the infection. Six days after inoculation with C. trachomatis, CYP1A- and CYP2B-mediated metabolism in the liver of the mice was diminished up to 49% of control levels. However, when animals were treated with N(G)-nitro-L-arginine methyl ester at days 4 and 6 postinfection, the decrease in the metabolism of CYP1A and CYP2B was largely blocked. These results suggest that C. trachomatis infection can depress cytochrome P450 in a manner similar to other types of infections and that NO is likely to be a mediator of this depression. This finding may be of significance to patients taking drugs that are metabolized by phase I enzymes during infections with some bacteria such as C. trachomatis.

Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.

Exposure to environmental tobacco smoke results in an increased production of (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide in juvenile ferret lung homogenates

Six-week-old ferrets were exposed head-only to clean air or environmental tobacco smoke (ETS) at an average particulate concentration of 38 +/- 13 mg/m3 for 2 h/d, 5 d/wk for up to 15 wk. Twenty four hours after last exposure, the ferrets were sacrificed and the metabolism of benzo[a]pyrene and (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol was studied in lung homogenates. The results show that after ETS exposure total metabolism of benzo[a]pyrene, measured by the accumulation of hexane nonextractable radioactivity, was increased by 35% in the males and 66% in the females (p < .05), respectively, of that observed with air-exposed controls. With (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol as substrate, the formation of both benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetraol and (+)-anti-benzo[a]-pyrene-7,8-dihydrodiol-9,10-epoxide-derived tetraols by lung homogenates of ETS-exposed male and female ferrets was significantly increased compared to the air-exposed controls (p < .01). DNA-bound radioactivity was significantly increased in both the males (p < .01) and females (p < .05) compared to the air-exposed ferrets.

Effect of hyperoxia on rat pulmonary and hepatic cytochrome P450 monooxygenases

Abstract Exposure of adult male rats to hyperoxia (O2 > 95%) resulted in a tendency for all of the components of the pulmonary cytochrome P450 (P450) system to increase at 48 h after the exposure. However, the most pronounced effect of hyperoxia was observed on pulmonary ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities which were induced 4- and 25-fold respectively after 48 h. In the liver, P450 and NADH b5 reductase were increased after 48 h, while other components of the monooxygenase system remained unchanged. In the hepatic microsomes, contrary to the lungs, aminopyrine N-demethylase activity was decreased after 24 h of hyperoxic exposure (P < 0.05) and returned to the control level by 48 h. Similar changes were observed in benzphetamine N-demethylase activity. Aniline hydroxylase activity was decreased after 8 h of hyperoxic exposure (P < 0.01) and remained decreased at 24 h (P < 0.01) and 48 h (P < 0.05). The level of induction of ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities, however, was almost similar in the liver to that observed in the lungs.

Undernutrition during hyperoxic exposure induces CYP2E1 in rat liver

Abstract Induction of cytochrome P450 2E1 (CYP2E1) has been shown to occur through two distinct mechanisms. The first is seen by treatment of rats with acetone, pyrazole, and 4-methyl-pyrazole, which induces CYP2E1 protein without affecting the mRNA level. The second is observed in starvation, diabetes, and obesity, in which an increase of CYP2E1 protein is associated with an increase of the CYP2E1 mRNA. It has been reported by (Tindberg and Ingelman-Sundberg 1989) that hyperoxic exposure (95% O2) induced a several-fold increase of CYP2E1 protein in both the liver and lung of exposed rats without affecting the level of CYP2E1 mRNA. During the course of our previous study which demonstrated hyperoxia-induced specific pretranslational induction of CYP1A1/2 in the liver and CYP1A1 in the lung, we observed a progressive increase of hepatic CYP2E1 mRNA in animals of the hyperoxia group. Hyperoxia is accompanied by some degree of starvation and our earlier experiments were conducted with rats of significantly greater body weight than those used by Tindberg and Ingelman-Sundberg (260 vs 150 g). Thus we reevaluated the changes of CYP2E1 in the current study with the use of food-restricted control, and by utilizing rats of comparable weight (∼150 g) to that utilized by Tindberg and Ingelman-Sundberg. The results obtained in the present study showed that there was a significant increase in the levels of hepatic CYP2E1 mRNA, protein, and p-nitrophenol hydroxylase activity in the food-restricted control group compared to the untreated controls. Rats from the hyperoxia group also demonstrated a similar increase of these three parameters in their livers but showed no significant difference compared with the results of the food-restricted control group. Rats weighing ∼260 g were also examined with similar food restriction and hyperoxia, and the results were essentially similar to those obtained with the younger rats. The lungs of rats from food-restricted control and hyperoxia groups showed no increase of any of the CYP2E1 parameters. The results obtained in the current study, therefore, indicate that hyperoxia has no effect on CYP2E1 expression in both the liver and lung. Increased CYP2E1 mRNA, protein, and p-nitrophenol hydroxylase activity seen in the liver of rats, but not in the lungs, are consistent with the notion that undernutrition during hyperoxia is the underlying mechanism for this induction.

Suppressive effect of interleukin-1 on pulmonary cytochrome P450 and superoxide anion production

Interleukin-1 has been shown to prolong the survival of rats exposed to lethal concentrations of oxygen. This oxygen tolerance has been attributed by some workers to an increase of manganese superoxide dismutase. We report here that the administration of interleukin-1 to male adult rats results in (i) significant decrease of pulmonary cytochrome P450 at 24 and 72 hours, (ii) decrease of P450 IIB1 mRNA at 24 and 72 hours and (iii) significant decrease of superoxide anion generation from pulmonary microsomes isolated from treated rats. To the best of our knowledge, this is the first report to demonstrate these effects of interleukin-1 on pulmonary P450 and its oxidase activity (O2- generation). On the basis of these results and several earlier reports in which various P450 depressants have been shown to depress superoxide production from microsomes and to prolong the lives of rodents in hyperoxia, we conclude that oxygen tolerance induced by interleukin-1 administration is likewise mediated, at least in part, by reduced generation of superoxide anion from cytochrome P450 monooxygenase system.