Octachloronaphthalene induction of hepatic microsomal aryl hydrocarbon hydroxylase activity in the immature male rat

Administration of octachloronaphthalene to immature male Wistar rats resulted in a dose-dependent increase in several enzymic, electrophoretic and spectral parameters associated with induction of the hepatic microsomal enzymes. Compared to corn-oil (control) treated animals octachloronaphthalene (150 μmol · kg^?1 induced hepatic cytochrome P-450 (1.5-fold), benzo [a]pyrene hydroxylase (18-fold) and 4-chlorobiphenyl hydroxylase (18-fold) enzyme activities. In addition to increases in the relative peak intensities of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide (EIC) difference spectra the peak maxima were observed at 448.5 and 452.2/428.0 nm, respectively. The effects of administering octachloronaphthalene to the rat were similar to those observed after pretreatment with 3-methylcholanthrene (MC) and electrophoresis of the induced microsomal proteins showed that both compounds enhanced heme-staining peptides with comparable electrophoretic mobilities. Moreover coadministration of MC (3 × 10 βmol · kg^?1) and octachloronaphthalene (2 × 150 μmol · kg^?1) indicated that their inductive effects were not additive. It was concluded that octachloronaphthalene was an MC-type inducer of hepatic microsomal enzymes.     

Increased aryl hydrocarbon hydroxylase activity in hepatic microsomes from streptozotocin-diabetic female rats.

In streptozotocin-induced diabetic male rats, hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity was depressed to less than control values, but was increased in microsomes from diabetic female rats. Insulin treatment of diabetic animals returned the altered AHH activity to control values in both sexes of rats. 2. Hepatic microsomal AHH activity was increased over control values in both sexes of diabetic mice. 3. Protection of female rats from the diabetogenic effects of streptozotocin by nicotinamide pretreatment also prevented the increase in AHH activity observed in unprotected animals. 4. Treatment of control and diabetic female rats with 3-methylcholanthrene resulted in larger increases in hepatic AHH activity in control animals, but similar increases in cytochrome P-448 content occurred in both treatment groups. 5. Differential stimulatory or inhibitory effects on AHH activity were observed after the addition of SKF 525-A, metyrapone, and rotenone to hepatic microsomes in vitro from control and diabetic female rats. However, similar stimulatory responses in AHH activity were observed after addition of alpha-naphthoflavone to microsomes from both treatment groups.     

Different levels of Schistosoma mansoni infection increased the mutagenicity of benzo(a)pyrene, the activity of aryl hydrocarbon hydroxylase and the formation of hepatic microsomal hydrogen peroxide

The present study investigates the influence of different levels of Schistosoma mansoni infection (60, 120, 180, 300, 600 cercariae per mice) after 33 days on the activity of aryl hydrocarbon hydroxylase (AHH) and the formation of hepatic hydrogen peroxide (H(2)O(2)) during the metabolic activation of benzo(a)pyrene [B(a)p]. Also, it shows the mutagenic effect of B(a)p at different levels of S. mansoni infection using Salmonella typhimurium TA 98 and TA 102 as a tester strains. High levels of H(2)O(2) production (222 nmol/mg protein) and AHH activity (240 pmol 3-OH B(a)p per mg protein) were seen at 300 cercariae per mice. Increasing histidine revertant colonies at TA98 and TA102 were detected at different levels of S. mansoni infection. These data clearly demonstrate that S. mansoni infection changes the mutagenicity of B(a)p, AHH activity, as well as enhancing the formation of hepatic H(2)O(2) generated during the metabolic activation of B(a)p in infected mice.     

Polycyclic aromatic hydrocarbon in vitro cytotoxicity to bluegill BF-2 cells: mediation by S-9 microsomal fraction and temperature

The in vitro cytotoxicities of various polycyclic aromatic hydrocarbons (PAHs) to bluegill sunfish BF-2 cells were determined with the neutral red assay, which was modified by the incorporation of an S-9 microsomal fraction. Whereas the PAHs per se were weakly cytotoxic, the presence of the S-9 fraction in the incubation mixture increased the cytotoxicity of many of the PAHs, presumably due to the formation of active metabolites. In addition, the cytotoxicity of the PAHs (whether in the absence or presence of the S-9 microsomal fraction) was potentiated if the 6 h exposure was at 37, rather than at 26 degrees C.     

Effect of scoparone on the hepatic microsomal UDP glucuronyltransferase activity in mice

The effect of scoparone on UDPglucuronyltransferase in mouse hepatic microsomes was studied. After treatment with scoparone, hepatic microsomal UDPglucuronyltransferase activity was increased with dose-dependent manner as compared to control. The V_max value (control=23.2 n moles/mg protein/min, scoparone=31.2 n moles/ mg protein/min) without affecting the K_m value (414 μM) for p-nitrophenol was increased by the scoparone treatment, and the pattern of kinetic studies for UDP-glucuronic acid was also similar to those of p-nitrophenol. Whereas, the hepatic microsomal UDPglucoronyltransferase was not changed by the addition of scoparone in vitro. The results obtained suggest that the characteristics of increase in the enzyme activity may include induction of enzyme proteins.     

Mutagenic activation of 2,4-diaminoanisole and 2-aminofluorene in vitro by liver and kidney fractions from aromatic hydrocarbon responsive and nonresponsive mice.

The mutagenicity of the two carcinogenic arylamines 2,4-diaminoanisole (2,4-DAA) and 2-aminofluorene (AF) was compared using liver and kidney fractions from two aromatic hydrocarbon (3-methylcholanthrene, MC) responsive and two nonresponsive mouse strains. MC pretreatment of mice caused an increase in 2,4-DAA mutagenicity with liver fractions from all four strains; however, much higher increases were seen in the two responsive than in the two nonresponsive strains. Kidney fractions had very low basal 2,4-DAA mutagenic activity. MC treatment led to 14–27-fold increase in 2,4-DAA mutagenicity in the responsive C57BL/6/BOM (B6) strain, but not in any of the other strains. AF mutagenicity was increased with liver fractions from all four mouse strains, but to the greatest extent in the B6 mice. AF showed high basal mutagenic activity with kidney fractions from all four strains, but MC treatment did not cause any increase in AF mutagenicity in any of the strains. Thus, there was a clear difference in the pattern of metabolic activation of the two arylamines 2,4-DAA and AF by liver and kidney fractions in mice, both with respect to constitutive activities and to the response to aromatic hydrocarbons.     

Correlations between polychlorinated biphenyl immunotoxicity, the aromatic hydrocarbon locus, and liver microsomal enzyme induction in C57BL/6 and DBA/2 mice

The suppression of the antibody response by polychlorinated biphenyls (PCB) in mice is dependent on the planarity of the PCB molecule and on the expression of the aromatic hydrocarbon (Ah) receptor. In this study, the hypothesis that this form of immunotoxicity is a consequence of the activation of the Ah gene complex and that other compounds which are Ah receptor ligands would also be immunotoxic was tested. 2,2',4,4'-Tetrachlorobiphenyl (TCB), 2,3,3',4,4',5-hexachlorobiphenyl (HCB), phenobarbital (PB), or beta-naphthoflavone (BNF) was given ip to either C57BL/6 (B6,Ahb/Ahb) or DBA/2 (D2, Ahd/Ahd) mice 2 days before immunization with sheep erythrocytes. Organ weights, histopathology, hemagglutinating antibody titers, and the splenic direct antibody plaque-forming cell (PFC) response were evaluated on Day 5. Hepatic aryl hydrocarbon hydroxylase (AHH) induction by these compounds and by 2,2',5,5'-TCB and 3,3',4,4'-TCB was measured as an indicator of Ah receptor binding and subsequent activation of the Ah gene complex by methylcholanthrene-type inducers, while aminopyrine N-demethylase (APND) was measured as an indicator of PB-type induction. 2,2',4,4'-TCB and PB had no effects on the immune parameters of either strain but induced APND activity in both strains. 2,2',5,5'-TCB slightly induced APND activity in B6 mice. 2,3,3',4,4',5-HCB caused a 70% suppression of PFC per spleen, decreased the serum antibody titer, elevated cytochrome P-450 levels (193%), induced both APND (165%) and AHH (217%) activity in B6 mice, but it induced only APND (156%) activity in D2 mice. 3,3',4,4'-TCB elevated cytochrome P-450 levels (210%) and induced both APND (129%) and AHH (321%) activities in B6 mice but only increased APND activities (115%) in D2 mice. BNF elevated cytochrome P-450 (144%), caused a 49% suppression in PFC per spleen, and induced both APND (156%) and AHH (248%) activities but only in B6 mice. These results support the hypothesis that the immunotoxicity caused by halogenated and polycyclic aromatic hydrocarbons is a consequence of activation of the Ah gene complex and suggests that this toxic effect can be initiated by any Ah receptor ligand.     

Genetically mediated induction of aryl hydrocarbon hydroxylase activity in mice by polychlorinated dibenzofuran isomers and 2,3,7,8-tetrachlorodibenzo-p-dioxin

Hepatic aryl hydrocarbon hydroxylase (AHH)-inducing potency of toxic polychlorinated aromatic hydrocarbons such as polychlorinated dibenzofurans (PCDFs), 3,4,5,3,4,5-hexachlorobiphenyl (HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in four inbred strains of mice with different phenotypes of Ah locus, i.e., AHH-responsive strains: C57BL/6N and AKR/Ms Qdj, and AHH-nonresponsive strains: DBA/2Cr Slc and Qdj; DDD. Eight individual PCDF isomers or TCDD were administered IP in doses of 30 g/kg; HCB was given in a dose of 120 g/kg. In AHH-nonresponsive strains of mice, only TCDD significantly induced hepatic AHH activity, while in AHH-responsive strains, 2,3,7,8-tetrachlorodibenzofuran(2,3,7,8-TCDF), 1,2,3,7,8-pentachlorodibenzofuran(1,2,3,7,8-PCDF) 2, 3, 4, 7, 8-pentachlorodibenzofuran (2, 3,4, 7, 8-PCDF), and TCDD significantly enhanced the enzyme activity, and the induced AHH activities with the three PCDF isomers were about 30–65% of those of TCDD. These results indicate that AHH responsiveness in mice segregates with the induction of AHH activity by PCDF isomers and may also segregate with the toxic potency of the isomers; i.e., toxic potencies of 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, and 2,3,4,7,8-PCDF in AHH-responsive strains of mice may be much greater than those in AHH-nonresponsive strains of mice. Taking into account both the potent AHH inducibility and the high bioaccumulation of 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, and 2,3,4,7,8-PCDF, these three PCDF isomers should be given greater attention with regard to environmental contamination.A part of this work was presented at the 41st annual meeting of the Japanese Society of Public Health, October 27–29, 1982, Fukuoka, Japan.     

Aryl hydrocarbon hydroxylase induction in mammalian liver-derived cell cultures. Effects of various metabolic inhibitors on the enzyme activity in hepatoma cells.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase induction in one mouse hepatoma and two rat hepatoma cell lines was characterized with respect to optimal growth requirements and optimal inducing concentrations of polycyclic hydrocarbons, phenobarbital, biogenic amines, and numerous other hydrophobic compounds. Cordycepin, ethidium bromide and puromycin aminonucleoside treatment of the mouse tumor line Hepa-1 produce strikingly different effects on the kinetics of hydroxylase induction, when the inducers benz[a]anthracene, phenobarbital and iso-proterenol are compared. without any of these ‘usual inducers’ present in the mouse hepatoma cell line, 0.50 to 5.0 mM puromycin aminonucleoside or 40 nM actinomycin-D causes large increases in the hydroxylase activity (at a time when gross RNA synthesis is virtually 100 per cent inhibited and gross protein synthesis is more than 50 per cent inhibited). This aminonucleoside- or actinomycin-D-mediated induction process induction, when the inducers benz[a]anthracene, phenobarbital and isoproterenol are compared. With-without prior treatment with the usual inducers and hence presumably without prior accumulation of putative induction-specific RNA: moreover, this induction process apparently requires little, or no. simultaneous RNA synthesis during exposure to the aminonucleoside or actinomycin-D. In contrast, when primary cultures derived from normal rat liver are treated with similar high concentrations of the aminonucleoside, no induction of the hydroxylase activity occurs. This result suggests that the effect of puromycin aminonucleoside on hydroxylase-specific mRNA synthesis, processing or stabilization is very different between the hepatoma cell line and normal fetal rat primary hepatocytes in culture.     

Correlation between in-vitro microsomal enzyme activity and whole organ hepatic elimination kinetics: analysis with a dispersion model

A new model, the dispersion model of hepatic elimination, is applied to the correlation between in-vitro microsomal data and corresponding rat isolated perfused liver data for a number of drugs reported in the literature, whose extraction ratio varies over the range of 0.01 to 0.995. The dispersion model described the data better than either the 'well-stirred' model or the 'parallel-tube' model, two other widely used models of hepatic elimination. The experimental data support the concept that elimination of solutes is affected by is a considerable dispersion on passage through the liver.     

Induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase and 2-acetylaminofluorene N-hydroxylase by polycyclic hydrocarbons in regenerating liver from inbred strains of mice

The effect of partial hepatectomy (67–75 per cent excised) on aryl hydrocarbon (benzo[a]pyrene) hydroxylase and 2-acetylaminofluorene N-hydroxylase activities in genetically responsive C57BL/6N and nonresponsive DBA/2N inbred strains of mice was studied. Basal aryl hydrocarbon hydroxylase activity in C57BL/6N mice is reduced by 70 per cent within 12 hr but returns to control values between 36 and 48 hr after the operation and remains at that level thereafter. Similar, but less pronounced, changes are observed in the DBA/2N mouse. Treatment with 3-methylcholanthrene (80 mg kg^?1 intraperitoneally) 1 hr after partial hepatectomy induces aryl hydrocarbon hydroxylase in C57BL/6N mice, by 48 hr, to the same extent as in mice with intact livers. In DBA/2N mice, even after partial hepatectomy, treatment with 3-methylcholanthrene has no effect on aryl hydrocarbon hydroxylase activity. The effect of partial hepatectomy and 3-methylcholanthrene treatment on 2-acetylaminofluorene N-hydroxylase activity in both strains of mice is similar to that observed on aryl hydrocarbon hydroxylase activity.     

Inhibition of morphine-induced analgesia and locomotor activity in strains of mice: a comparison of long-acting opiate antagonists

The long-acting opiate antagonistic potency of naloxazone (NXZ), beta-chlornaltrexamine (beta-CNA) and beta-funaltrexamine (beta-FNA) was compared using three inbred strains of mice, in which morphine induces either analgesia (DBA/2), locomotion (C57BL/6), or both responses (C3H/He). The antagonists were applied SC 24-120 hr before morphine (10 or 20 mg/kg, IP), followed by the tests after 30 min. The minimal dose which completely antagonized morphine-induced analgesia in DBA and locomotion in C57 mice during 24 hr were: for NXZ 50 and 100 mg/kg, for beta-CNA 0.8 and 6.2 mg/kg, for beta-FNA 1.6 and 12.5 mg/kg, respectively. beta-FNA and beta-CNA more potently blocked morphine-induced analgesia in DBA mice than the activity response in the C57 strain. In contrast, beta-FNA prevented morphine-induced locomotion at a lower dose (6.2 mg/kg) than analgesia (greater than 50 mg/kg) in C3H mice, while beta-CNA was equipotent (1.6 mg/kg). In general, beta-CNA turned out to be the most reactive compound, antagonizing morphine effects in low doses up to 120 hr. beta-FNA selectively antagonized either morphine-induced analgesia or locomotion, depending on the strain used. This suggests that a given morphine response might be caused by a genetically determined multiplicity of opiate receptor types and their mutual interactions.     

Glutathione S-transferase activity from guinea-pig brain: a comparison with hepatic multiple forms

1. Specific activities of glutathione S-transferase towards four model substrates were determined in guinea-pig brain 50,000 g supernatant and compared with those obtained for liver and kidney extract. 2. By using 1-chloro-2,4-dinitrobenzene as substrate, glutathione S-transferase activity was measured in different anatomical regions of brain; cerebellum expressed the highest conjugating capacity. 3. Brain glutathione S-transferase was resolved into four major peaks (PI 6.10, 6.60, 7.15, 7.65) each having similar kinetic constants for both substrates GSH and 1-chloro-2,4-dinitrobenzene. 4. Likewise, four forms, focused at pH 6.45, 7.14, 7.50 and 8.88, were obtained from liver. 5. Unlike hepatic tissue, brain does not possess the highly alkaline form which displays Se-independent GSH peroxidase activity. 6. Several psychotropic agents, including chlorpromazine and chlorazepate, produced a considerable in vitro inhibition on brain transferase activity.