Beta NF Research Articles

Cytochrome P450isoenzyme activities in cultured rat and mouse liver slices

1. The objective of this study was to determine the basal and inducible activities of several cytochrome P450 (CYP) isozymes and monitor the acinar and hepatocyte morphology in precision cut, cultured rat and mouse liver slices. 2. The slices were cultured up to 96 h in Chee's essential medium supplemented with insulin, transferrin, selenium, DMSO, dexamethasone and epidermal growth factor. A dynamic roller system was used to incubate the slices at 37 C in an atmosphere of 95% O2 :5% CO2. 3. Histopathology of the liver slices revealed maintenance of normal hepatic lobular architecture with time in culture. 4. CYP isozyme activities were measured at various times of culture. In rat liver slices, at72 h, CYP1A1 1A2 activity was induced 4-fold by beta NF and 37-fold by dioxin (TCDD) whereas in mouse liver slices, 1A1 1A2 activity was not inducible by beta NF but was induced 19-fold by TCDD. At 72 h, CYP2A5 (coumarin-7-hydroxylase) activity was not detected in rat liver slices but in mouse liver slices, 2A5 was induced 2-fold by beta NF, 11-fold by phenobarbital (PB) and 3-fold by TCDD. 5. Hydroxylation of testosterone at specific positions was used as an indication of the activities of various P450 isoforms. Testosterone was added to the cultures at 0 and 72 h and the metabolites were measured at 24 and 96 h respectively by hplc analysis. Depending upon the species, the treatment and the time in culture, CYP1A, 2A, 3A, 2B and 2C activities were detectable. 3A activity was highly induced by PB in both rat and mouse liver slices. These results demonstrate that this culture system can be used to assess and compare xenobiotic metabolism in liver slices from rodent species.

Strain-specific enhancement or inhibition of coumarin hepatotoxicity in mice following pretreatment with two different liver enzyme-inducing agents.

Human exposure to coumarin continues despite controversy over its hepatotoxic potential. Greater understanding of human reactions to coumarin may be achieved by studying murine interstrain differences. The metabolic basis of coumarin hepatotoxicity and its modulation by liver enzyme inducers, beta-naphthoflavone (beta NF) and aroclor 1254 (ARO), were investigated in C3H/He and DBA/2 mice. Coumarin (200 mg/kg) was hepatotoxic to both strains, resulting in 2- to 15-fold plasma aminotransferase elevations, mild subcapsular linear hepatocyte necrosis after 24 hr, and, in some C3H/He mice, centrilobular necrosis. In this strain, beta NF pretreatment caused a 2- to 3-fold further increase in plasma aminotransferases and produced periportal necrosis. In contrast, ARO-pretreated C3H/He mice tended to exhibit lower plasma aminotransferases and occasional midzonal damage. Neither pretreatment significantly altered coumarin hepatotoxicity in DBA/2 mice. In C3H/He mice, hepatic microsomal metabolism of [3-14C]-coumarin via the 3-hydroxylation pathway doubled following both beta NF and ARO treatment. The contrasting nonresponsiveness of DBA/2 mice suggested that this pathway is linked to the Ah locus, which is defective in this strain. ARO treatment caused a maximal 5-fold increase in coumarin 7-hydroxylation in C3H/He mice, whereas DBA/2 mice were 30% less responsive. Potentiation of coumarin hepatotoxicity corresponded to an increase in the 3-:7-coumarin hydroxylation ratio. Pretreatment-dependent shifts in the location of hepatocyte damage may be related to changes in the translobular ratio of enzymes involved in activation and detoxication of coumarin. These data highlight how genetic background, individual variation, and xenobiotic-induced alterations in enzyme profiles, factors all relevant to human risk assessment, can influence the consequence of coumarin exposure.

Monooxygenation, cytochrome P4501A1 and P4501A1-mRNA in rat liver slices exposed to beta-naphthoflavone and dexamethasone in vitro

Precision-cut liver slices (0.5 mm) were incubated at 30 degrees C in a modified William's Medium E for up to 48 hrs. During the incubation, K+ and GSH/GSSG concentrations did not decrease. Cytochrome P450-dependent dealkylation rates of 7-ethoxycoumarin (ECOD), 7-allyloxycoumarin (ACOD) and 7-ethoxyresorufin (EROD) decreased to 1/3, 1/2 or did not change at all, respectively, after a 48 hrs incubation period. Exposure of the slices to 25 microM beta-naphthoflavone (beta NF) resulted in about 3 times higher monooxygenation rates. An exposure to a combination beta NF and dexamethasone (10(-6)M) caused a marked induction (6 times higher rates) after 48 hrs. Simultaneously an increase in P4501A1 content was observed. P4501A1-mRNA expression (measured by RT-PCR) was distinctly increased following beta NF exposure for 6 or 24 hrs. DMSO (0.2%) and dexamethasone alone modified monooxygenation rates, but did not have significant effects on P4501A1 content or, in the case of DMSO, P4501A1 gene expression (for dexamethasone not determined). Liver slices are a useful and simple tool for the detection of a beta NF-like induction within a few hours after preparation of the slices.

Assessment of Regional Cytochrome P450 Activities in Rat Liver Slices Using Resorufin Substrates and Fluorescence Confocal Laser Cytometry

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Variation in the response of T cells to concanavalin A after in vitro exposure to benzo[A]pyrene and 2-aminofluorene.

The ability of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BP) and its metabolites to be immunosuppressive has been well documented by many investigators. The arylamine, 2-aminofluorene (AF) and its metabolic intermediates have not been as widely studied in this regard. Here, we investigate the effect of BP, 3-hydroxy-BP (3-OH-BP), AF, N-hydroxy-AF (N-OH-AF) and acetyl-AF (AAF) on T-cell proliferation using the T-cell mitogen, Concanavalin A (ConA). These compounds as well as BP-7, 8-diol-9, 10-epoxide (BPDE) were also used to determine their effect on T-cell-mitogen binding. Both AF and BP are substrates for the P-450 and flavin-containing monooxygenase enzyme system, which can be induced with beta-naphthoflavone (beta NF). We incubated beta nF with BP and AF to determine the effect of a P-450 inducer on BP and AF mediated-ConA suppression. Here we demonstrate that BP, 3-OH-BP, AF, and AAF are able to suppress the proliferative response to ConA, while N-OH-AF cannot. Further, we show that BP, 3-OH-BP, BPDE, AF and N-OH-AF do not alter the ability of ConA to bind the mitogen receptor of splenic T-cells, indicating an intracellular mechanism for suppression. Studies with beta NF indicate that this P-450 inducer enhances the anti-proliferative effect of BP, while it abolishes this effect of AF.

Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol.

1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -CH3, -C2H5, -iC3H7) have been determined with beta-naphthoflavone (beta NF)-induced rat liver microsomes and produced reverse type I spectral changes. Ks,app varied from 0.14 mM for 3,5-diiC3H7-paracetamol to 2.8 mM for paracetamol. 2. All seven analogues underwent rat liver microsomal cytochrome P450-dependent oxidation, as reflected by the formation of GSSG in the presence of GSH. The GSSG-formation was increased in all cases upon pretreatment of rats by beta-naphthoflavone (beta NF) and was generally decreased upon pretreatment by phenobarbital (PB). 3. Rat liver microsomal cytochrome P450 as well as horseradish peroxidase catalysed the formation of 3,5-disubstituted NAPQI analogues from the corresponding parent compounds, as identified by UV-spectrophotometry of the NAPQI analogues and by GC/MS detection of the following GSH-conjugates: 2-glutathione-S-yl-3,5-dimethyl-1,4-dihydroxybenzene, 2-glutathione-S-yl-3,5-dichloro-paracetamol, and 2-glutathione-S-yl-3,5-dibromo-paracetamol. 4. In liver microsomal (beta NF-induced) incubations, apparent K(m) values, as determined for the cytochrome P450 catalysis-dependent oxidation of GSH, for seven 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -CH3, -C2H5, iC3H7) varied from 0.07 to 0.64 mM. Paracetamol exhibited an apparent K(m) of 0.73 mM. Apparent Vmax values for the cytochrome P450 catalysis dependent oxidation of GSH varied from 0.66 nmol min-1 mg-1 protein for paracetamol to 3.0 nmol min-1 mg-1 protein for 3,5-dimethyl-paracetamol.

In vitro biotransformation of aflatoxin B1 (AFB1) in channel catfish liver.

The biotransformation of the dietary carcinogen aflatoxin B1 (AFB1) was examined in hepatic microsomal and cytosolic fractions from channel catfish, an aquatic species shown to be refractory to AFB1 toxicity and reported to be resistant to AFB1 hepatocarcinogenesis. Catfish liver microsomes catalyzed the in vitro oxidation of AFB1 to the reactive AFB1-8,9-epoxide (AFBO) at high substrate concentrations (128 microM AFB1) but not at low substrate concentrations (16 microM AFB1) which were more representative of environmental exposure. A similar trend was observed for the production of the hydroxylated metabolite aflatoxin M1 (AFM1). In contrast, hepatic microsomes prepared from rainbow trout, a species sensitive to AFB1 toxicity and hepatocarcinogenesis, activated AFB1 at 16 and 128 microM AFB1, with the rate of AFB1 epoxidation by trout microsomes exceeding that of catfish by more than sixfold. Treatment of channel catfish with 5,6-benzoflavone (beta NF, 20 mg/kg) resulted in a threefold increase in AFM1 formation but did not affect AFBO formation. AFB1 was rapidly reduced to aflatoxicol (AFL), a putative detoxification product of AFB1, at both low and high substrate concentrations. The rate of AFL production by channel catfish hepatic cytosol was 40- and 65-fold greater than observed for rainbow trout at 16 and 128 microM AFB1, respectively. Western blotting of catfish cytosols revealed the presence of a catfish cytosolic protein of approximately 25 kDa that displayed immunological cross-reactivity to rat GST Ya, but not to other rat or mouse alpha class GSTs with high AFBO-conjugating activity. Furthermore, catfish cytosolic GSTs did not catalyze the conjugation of AFBO with GSH. The results of these studies indicate that AFB1 is poorly oxidized by channel catfish microsomes, and suggest that the lack of microsomal AFB1 activation together with the rapid conversion of AFB1 to AFL contributes to the apparent resistance of channel catfish to AFB1 toxicity and hepatocarcinogenesis.

Effects of inhibition and induction of cytochrome P-450 isozymes on hyperoxic lung injury in rats.

Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1, the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. beta-Naphthoflavone (beta NF), a selective inducer of cytochrome P-450IA1, was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IA1 activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of beta NF, a small increase in survival time was found from 71.1 +/- 8.7 to 88.0 +/- 20.2 h; however, there was no difference in the induction of cytochrome P-450IA1 activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of beta NF is thus unrelated to cytochrome P-450IA1 induction. We conclude that neither inhibition of cytochrome P-450IIB1 activity nor induction of cytochrome P-450IA1 activity protects adult rats against hyperoxic lung injury.

Long-Term (Imprinting) Effects of Transplacental Treatment of Mice with 3-Methylcholanthrene or β-Naphthoflavone on Hepatic Metabolism of 3-Methylcholanthrene

Foetal mice of genotype AhbAhd (responsive to induction of metabolism of polycyclic aromatic hydrocarbons [PAH]) or AhdAhd (non-responsive) were exposed transplacentally on gestation day 17 to a single dose of 3-methylcholanthrene (MC, 5-175 mg/kg) with or without prior treatment on day 15 with beta-naphthoflavone (beta NF, 150 mg/kg). The mothers were themselves either induction-responsive [(C57BL/6 x DBA/2)F1] or non-responsive (DBA/2). Metabolism of [14C]MC by homogenates of livers from the transplacentally-exposed offspring was quantified at 9 months of age (first experiment) or 13 months (second experiment) with or without prior inducing treatment with MC. The foetal exposure to MC had a permanent effect on MC metabolism by the adult hepatic homogenates in both experiments. In most instances the effect was positive in direction and small in magnitude (15-30%). It was dose-dependent with regard to transplacental MC, occurred in both induced (AhbAhd) and non-induced (AhdAhd) individuals, and was significant only when the mother and/or the foetus was inducible. beta NF itself did not have a positive imprinting effect. In some cases it either reduced or potentiated the long-term imprinting effect of MC, depending on the MC dose and the phenotype of the mother. These results confirm that transplacental exposure to a carcinogenic PAH may permanently alter metabolism of the chemical in later life, and indicate that this imprinting action is dependent on induced metabolism of the chemical in the mother and/or foetus.

Gene Expression, Ontogeny and Transplacental Induction of Hepatic UDP-Glucuronosyl Transferase Activity in Mice

The ontogeny and transplacental inducibility of UDP-glucuronosyl transferase (UDPGT) activities potentially relevant to detoxification of polycyclic aromatic hydrocarbons were studied in (C57BL/6 x DBA/2) F1 or (DBA/2 x C57BL/6) F1 fetal mouse liver, with p-nitrophenol (PNP) and 3-hydroxybenzo[a]pyrene (3-OH-BP) as substrates. Both UDPGT activities developed during the late fetal period and reached almost 60% of the adult activity at term; PNP, but not 3-OH-BP UDPGT decreased significantly on postnatal day 1 before rising to adult levels. A single exposure to beta-naphthoflavone (beta NF; 150 mg/kg) on day 17 of gestation induced the PNP-UDPGT activity significantly (1.5-fold) by day 19 in the B6D2 F1s but not D2B6 F1s. A single dose of 3-methylcholanthrene (MC; 100 mg/kg) or 2,3,7,8,-tetrachlorodibenzo-p-dioxin (10 micrograms/kg) did not induce, but three injections of MC also resulted in significant induction in the fetuses of C57BL/6 mothers. 3-OH-BP-UDPGT was not significantly induced by any of the chemicals in either genetic cross. In a parallel study, a gene for an inducible mouse UDPGT, designated UDPGTm-1, was shown by Northern blotting to be expressed in fetal liver by day 18 of gestation at low levels relative to adults, but was not induced transplacentally by MC, beta NF or phenobarbital (PB). These results show that (1) at least two functionally defined UDPGT activities toward phenolic substrates are present in the late fetal mouse liver; (2) one of these is transplacentally inducible by beta NF and MC, but only in fetuses of C57BL/6 mothers, (3) induction where achieved was relatively small in magnitude, and (4) a gene of a PB-inducible UDPGT was expressed at low levels in the fetuses but was not induced transplacentally.

Mutagenicity of 3‐chlorodibenzofuran and its metabolic activation

3-Chlorodibenzofuran was the only markedly mutagenic isomer among the four monochlorodibenzofurans. Although it was mutagenic even in the absence of 9,000g supernatant fraction (S9) of rat liver, it was further activated by the addition of S9. Metabolic activation of this compound in mutagenicity was studied using liver S9s and cell fractions which were prepared from rats treated with two inducers. 1,1-Dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) was used as an inducer of phenobarbital inducible cytochrome P-450, and beta-naphthoflavone (beta NF) was used as an inducer of 3-methylcholanthrene inducible cytochrome P-448. S9, microsomal, mitochondrial, and cytosolic fractions were obtained from four groups of rats, i.e., untreated, DDE treated, beta NF treated, and DDE and beta NF treated groups. Mutagenicity was tested using Salmonella typhimurium tester strain TA98, because this strain is more sensitive to 3-chlorodibenzofuran than strain TA100. This experiment showed that 3-chlorodibenzofuran was activated most highly by beta NF-induced microsomes. However, it was also activated by the cytosolic fraction. Moreover, it was highly activated in rat livers which were not treated with inducers. The activity of aryl hydrocarbon hydroxylase (AHH) of each fraction was measured. AHH did not always become an index of the metabolic activation of 3-chlorodibenzofuran. This study showed that 3-chlorodibenzofuran is activated not only by cytochrome P-448, which is induced by 3-methylcholanthrene type inducers, but also by the enzymes existing in normal rat liver. This result suggests a risk of manifestation of its toxicity to normal animals.

Effect of pretreatment with β-naphthoflavone on tumorigenesis by N-nitrosoethylurea in five mouse strains

The non-carcinogenic inducer of the Ah locus, beta-naphthoflavone (beta NF), was administered to females of 5 mouse strains at a dose of 150 mg/kg 48 h before treatment with a tumorigenic dose of the direct-acting carcinogen, N-nitrosoethylurea (ENU), once weekly for 4 weeks. The strains used were C57BL/6, C3H/He and NIH Swiss (responsive to Ah locus induction) and AKR and DBA/2 (induction-non-responsive). The ENU caused primary lung tumors in all strains and in some cases smaller numbers of other neoplasms, including lymphomas, sarcomas and hepatocellular tumors. The beta NF pretreatment did not reduce the numbers of any of the tumors, compared with mice given ENU alone. This result is in contrast to previous findings of a strong protective effect of beta NF against tumorigenesis in Ah-responsive strains by the metabolism-dependent carcinogens, benzo[a]pyrene and 3-methylcholanthrene and confirms that this protection is directly related to enzyme induction. beta NF treatment caused a significant doubling in the number of lung tumor bearers among the ENU-exposed C57BL/6 mice but in no other strain, suggesting the possibility of strain-specific tumor promotion.

3,5-Diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine inactivates rat liver cytochrome P-450c, but not its orthologue, rabbit lung form 6

Various rat liver cytochrome P-450 (P-450) isozymes are targets for mechanism-based inactivation by 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (4-ethyl DDC). Unlike rat liver, which contains multiple P-450 isozymes, rabbit lung contains only three major isozymes referred to as forms 2, 5, and 6. We have examined the ability of 4-ethyl DDC to destroy P-450 heme in hepatic and pulmonary microsomes from untreated and beta-naphthoflavone (beta NF)-treated rabbits. This compound destroyed 31% of the P-450 in either hepatic microsomal preparation, but was ineffective at lowering P-450 and heme levels in pulmonary microsomes when examined at a range of concentrations (0.45-5.0 mM). These data suggest that rabbit pulmonary P-450 forms 2, 5, and 6 are not targets for destruction by 4-ethyl DDC, despite the ability of this compound to inactivate rat liver P-450c, the orthologue of rabbit lung form 6.

The 4S polycyclic aromatic hydrocarbon-binding protein: immunohistochemical localization in mice

The 4S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a cytoplasmic protein that binds PAHs with specificity and high affinity. We have used antisera for the PBP and unlabeled peroxidase anti-peroxidase immunohistochemistry to demonstrate its possible localization in cell types known to have xenobiotic metabolizing capabilities. Cellular sites of the PBP in liver, lung and kidney of C57BL/6 and DBA/2 mice were probed. The PBP was visualized in hepatocytes throughout the liver lobule and was not preferentially located in either centrilobular or periportal areas. However, cellular heterogeneity with respect to PBP content was clearly evident in the hepatocyte population. The positive reactivity correlated with substantial levels of benzo[a]pyrene (B[a]P) binding in liver cytosol. In the lung, the PBP was found in the bronchiolar epithelium and the alveolar septa, and was localized in ciliated and non-ciliated Clara and alveolar type II cells as well as in alveolar macrophages. In the kidney, the glomeruli and epithelia of proximal and distal convoluted tubules and collecting ducts were labeled. Staining for the PBP was greatest in the apical region of the pyramid and was localized in the epithelial lining of the collecting ducts. Relatively lower levels of the PBP were detected in the lung and kidney than in the liver. Staining was localized in the cytoplasmic compartment of cells in all tissues examined. Similar immunoreactivities were exhibited in the tissues of both C57BL/6 and DBA/2 mice. Treatment with beta-naphthoflavone (beta NF) altered neither the intensity nor pattern of immunostaining. Furthermore, treatment with beta NF or isosafrole has no effect on the Kd and Bmax of B[a]P binding to liver cytosolic PBP. The results of our experiments demonstrate localization of the PBP to sites of active physiological response to PAH exposure.

Role of the maternal environment in determining susceptibility to transplacentally induced chemical carcinogenesis in mouse fetuses

Treatment of pregnant mice with 3-methylcholanthrene (MC) causes lung and liver tumors in the offspring, the incidences of which are greatly influenced by the Ah locus regulated induction phenotype for aryl hydrocarbon hydroxylase activity (AHH) in both the mother and fetuses. In order to examine the biochemical and molecular mechanisms responsible for the modulating effect of maternal environment on tumor susceptibility, reciprocal crosses between responsive C57BL/6 and non-responsive DBA/2 mice were made and the pregnant mothers were treated i.p. on the 17th day of gestation with either olive oil alone, 30 mg/kg of MC, or 30 mg/kg of beta-naphthoflavone (beta NF). At various times after injection, the mothers were killed and the fetuses removed for enzymatic and molecular blot analysis. In fetal lung tissues, the absolute levels and relative induction ratios of AHH activity from D2B6F1 fetuses were very similar to those obtained in B6D2F1 fetuses during the first 24 h following a transplacental exposure to either inducing agent. This was also the case 48 h after an injection of beta NF. However, 48 h after exposure to MC, the AHH activity in fetal lungs from B6 mothers had declined to practically control values, whereas fetal lungs from D2 mothers still exhibited a high level of AHH activity. Similar induction kinetics for the CYPIA1 gene were obtained in fetal livers. These results were confirmed at the RNA level by quantitative slot-blot analysis of fetal RNA preparations. In both organs, treatment with inducing agents for the P450IA1 gene resulted in a rapid and early induction of CYPIA1 RNA by 4 h. Fetuses from D2 mothers, however, showed a more sustained induction of CYPIA1 RNA following exposure to MC than did fetuses from B6 mothers. These results suggest that the observed increase in tumor susceptibility observed in the offspring of D2 mothers compared to the offspring of B6 mothers was due, at least in part, to the differences in the persistence of induction of the CYPIA1 gene locus, and may be the result of differences in the clearance rates of MC from the fetal and maternal compartments or its pharmacokinetic distribution in the two types of maternal environments.

Effect of beta-naphthoflavone on o-tolyl saligenin phosphate-induced delayed neuropathy in two lines of chickens.

The effect of the microsomal enzyme inducer beta-naphthoflavone (beta NF) on the development of organophosphorus-induced delayed neuropathy (OPIDN) was examined in two laboratories (VPI and MSU), utilizing two strains of White Leghorn hens. A single intraperitoneal injection of beta NF at 80 mg/kg body weight 48 h prior to administration of o-tolyl saligenin phosphate (TSP), the neuroactive metabolite of tri-o-tolyl phosphate (TOTP), caused a significant increase in hepatic microsomal cytochrome P-450 concentrations and aniline hydroxylase activities after 72 h in both strains. Hepatic carboxylesterase and cholinesterase activities were not affected by beta NF treatment in either strain. Administration of TSP in single subcutaneous doses of 20 and 25 mg/kg body weight (VPI) or 30 and 60 mg/kg body weight (MSU) caused significant inhibition of whole-brain neuropathy target esterase (NTE) activity 24 h postdosing, and hens subsequently developed clinical signs characteristics of OPIDN. beta NF had no significant effect on NTE inhibition or on initiation or severity of OPIDN clinical signs. However, OPIDN clinical signs were less severe in the strain of bird (MSU) with the higher intrinsic hepatic carboxylesterase activity and the higher beta NF-induced cytochrome P-450 concentration. The study indicates that microsomal enzyme induction, which has been shown to alleviate TOTP-induced delayed neuropathy, could not alleviate OPIDN resulting from exposure to TSP. This study also suggests that strain may affect susceptibility to TSP-induced delayed neuropathy.

The use of single-sample clearance estimates to probe hepatic drug metabolism in rats. IV. A model for possible application to phenotyping xenobiotic influences on human drug metabolism.

1. Conditions were examined under which estimates of drug clearance made from a single measurement of plasma concentration effectively represented multi-sample estimates of clearance for carbamazepine, quinidine, and paracetamol. When plasma concentrations were measured at various post-dose times, both individual and mean values of single-sample clearance estimates (CL) corresponded closely to multi-sample clearance estimates. Best post-dose sampling times were: carbamazepine, 3 h; quinidine, 10 h; and paracetamol, 6 h. 2. Single sample clearance estimates, CL, were calculated for seven drugs employed as probes of hepatic drug-metabolizing activity in rats. Valproic acid was investigated as a probe of microsomal and peroxisomal oxidases; antipyrine, theophylline, ethosuximide, carbamazepine and quinidine as probes of hepatic mixed-function oxidases (MFO), and paracetamol as a probe for UDP-glucuronosyltransferase activity. 3. A clearance index (CI, namely, probe CL after xenobiotic pretreatment divided by control probe CL) was calculated for each probe. Eight pretreatments were used: phenobarbital (PB), beta-naphthoflavone (beta NF), polychlorinated biphenyls (PCB), rifampin, pregnenolone-16 alpha-carbonitrile (PCN), clofibric acid, cimetidine, and piperonyl butoxide. The effect of each xenobiotic pretreatment on all probe CL values was consolidated and plotted as the logarithm of the CI, and a distinct pattern or handprint evolved for each pretreatment. 4. We conclude that the use of multiple single-sample probes of hepatic MFO activity can be useful in structuring handprints to characterize xenobiotic-mediated effects on hepatic MFO. This minimally invasive in vivo approach may have application in investigating and possibly phenotyping MFO activity in human subpopulations that are subject to sustained exposure to particular xenobiotics.

Alteration of methylcholanthrene-mediated suppression of cutaneous delayed hypersensitivity by benzoflavone treatment of C57BL/6J mice

The potential role of the cytochromes P-450 in methylcholanthrene (MC)-mediated suppression of cutaneous delayed hypersensitivity (CDH) in C57BL/6J (B6) mice was evaluated indirectly by treating mice with agents known to induce or inhibit hepatic cytochromes P-450 prior to contact sensitization. Subsequent alterations in aryl hydrocarbon hydroxylase (AHH) activity and CDH, as measured by suppression of 2,4-dinitrofluorobenzene (DNFB)-induced ear swelling, were measured. MC administration resulted in a dose-dependent suppression of ear swelling and a concomitant dose-dependent induction of hepatic AHH activity. Treatment of B6 mice with phenobarbital (PB), 80 mg/kg daily X 3, a broad spectrum inducer of P-450, resulted in a 2.5-fold increase in benzo[a]pyrene (B[a]P) hydroxylase activity without affecting CDH. Animals treated with the same PB protocol prior to an ED20 dose of MC showed no difference in suppression of CDH compared to animals treated with MC alone. In contrast, successive treatment with the selective P1-450 inducer, 5,6-benzoflavone (beta NF), prior to and following an ED20 dose of MC significantly increased suppression of CDH (p less than 0.001) usually seen at this MC dose. Treatment with a known inhibitor of cytochrome P1-450 activity, 7,8-benzoflavone (alpha NF), did not prevent AHH induction when administered prior to and following MC (ED100) nor did it suppress CDH when administered alone. However, this alpha NF treatment completely prevented suppression of CDH usually seen at this MC dose. These data provide evidence suggesting that metabolic activation by cytochrome P1-450 is required for the expression of the immunosuppressive activity of MC.

Protection of rat from oxygen toxicity by inducers of cytochrome P-450 system.

Rats were pretreated with various inducers of cytochrome P-450 before being exposed to pure normobaric oxygen (O2) in order to determine whether the inducers interfere with toxicity. The pulmonary and liver inducers beta-naphthoflavone (beta NF) and 3-methylcholanthrene (3MC) increased the survival rate and decreased the amount of pleural and lung fluid accumulation in adult rats exposed to oxygen. Phenobarbital (PB), which is essentially active in the hepatic microsomal cytochrome P-450, was less effective in counteracting oxygen toxicity. After 7 days of exposure to oxygen, none of the untreated rats survived, whereas 40, 73, and 90% survival was observed in rats treated with PB, 3MC, and beta NF, respectively. After 60 h of O2 exposure, significantly less pleural and lung fluid accumulation was observed in beta NF- and 3MC-treated rats than in untreated or PB-treated rats (p less than 0.001). Both beta NF and 3MC prevented the increase of lung peroxidation (assessed by measuring of malondialdehyde) and that of hydrogen peroxide production by lung microsomes induced by O2 exposure. These protective effects are associated with a large increase in the components of the pulmonary cytochrome P-450 system and its peroxidase activity and with an increased response to hyperoxia by lung antioxidant enzyme activities. In contrast, in control rats, the activities of the antioxidant enzymes were not increased, and both the quantity and the peroxidase activity of cytochrome P-450 were significantly decreased by O2 exposure. We conclude that in the rat, pretreatment by inducers of pulmonary cytochrome P-450 results in marked protection against O2 toxicity and an increase of antioxidant enzyme response to hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS)