Nonadditive interactive effects of polychlorinated biphenyl congeners in rats: role of the 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor

Administration of 3,3',4,4',5,5'-hexa-,3,3',4,4',5-penta-, and 2,3,3'4,4'5-hexa-chlorobiphenyl to immature male Wistar rats caused a thymic atrophy at high dose levels (1.25, 1.0, and 100 mumol/kg, respectively) and induced the hepatic cytochrome P-448 dependent monooxygenases (benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase) at both high and low (0.25, 0.01, and 5 mumol/kg, respectively) doses. In contrast, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) (300 mumol/kg) did not elicit any of these effects but elevated hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein levels (threefold) as previously reported. The effects of hepatic receptor modulation by 2,2',4,4',5,5'-HCBP (300 mumol/kg) on the enzyme induction activities of 3,3'4,4',5-penta-, 3,3'4,4',5,5'-hexa-, and 2,3,3',4,4',5-hexa-chlorobiphenyl were dose-dependent; no interactive effects were observed at high (toxic) doses of these compounds, whereas apparent synergistically increased hepatic microsomal monooxygenase induction activities were noted at the lower submaximal induction doses. It was concluded that the increased responsiveness of the rats was due to elevated hepatic 2,3,7,8-TCDD receptor levels.     

Induction of cytochrome P-450 isozymes by mirex and chlordecone

The effect of the insecticides, mirex and chordecone (Kepone), on the cytochrome P-450 monooxygenase system in C57BL/6N mouse liver microsomes was studied. Mice were treated intraperitoneally with low (6 mg/kg) and high (30 mg/kg) doses of mirex and chlordecone in corn oil for 2 days. For comparison, mice were also treated with either phenobarbital (PB) or 3-methylcholanthrene (3-MC). All treatments significantly increased the hepatic microsomal P-450 content over that of controls. Benzphetamine N-demethylase, ethoxyresorufin O-deethylase, benzo[a]pyrene hydroxylase, and acetanilide hydroxylase activities were also determined. Mirex and chlordecone resembled phenobarbital with respect to the induction of monooxygenase activities. Immunoquantitation with antibodies to purified P-450 IIB1 (Pb-induced P-450) and P-450 IA1 (3-MC-induced P-450) indicated that mirex and chlordecone induced P-450 IIB1 in a dose-dependent manner. The high dose of mirex also induced a small amount of a protein cross reacting with the antibody to IA1. The induction of this isozyme did not, however, contribute significantly to the monooxygenase activities measured.     

Effects of inducer pretreatment on liver function and morphology in the mountain vole Microtus montanus

Liver function and morphology of the mountain vole, Microtus montanus, were examined after i.p. injections of phenobarbital, beta-naphthoflavone, or Aroclor 1254 at three dose levels. The results of the liver function tests showed serum glutamic pyruvic transaminase and serum malathion carboxylesterase activities were normal in all the treatment groups. The histological results showed no necrotic tissue but did reveal two different morphological stages related to the level of monooxygenase activity; a low induction state was represented by foamy vacuolated hepatocytes while high induction states were related to enlarged, swollen, hypertrophied cells.     

Induction in rainbow trout of an acute phase (C-reactive) protein by chemicals of environmental concern

1. Rainbow trout, Salmo gairdneri, produce elevated amounts of a serum acute phase (C-reactive) protein (CRP) when administered a variety of chemicals of environmental importance. 2. Compounds administered in doses which induce the cytochrome(s) P450 catalytic enzymes in trout hepatic microsomes also induce serum CRP. 3. However, an interferon-inducing virus does not induce CRP. Interferon induction by the virus is not significantly inhibited by chemicals which induce trout cytochrome(s) P450. 4. Simultaneous administration of chemicals and virus or virus alone results in depression of P450 protein production and only minor induction of CRP. 5. Thus, as with mammals, a reciprocating relationship appears to exist between the hemeprotein monooxygenase and immune systems of this freshwater teleost, and C-reactive protein appears to fit the reciprocating scheme closer to the cytochromes P450 response.     

Suppression of the inductive effects of phenobarbital and 3-methylcholanthrene on ascorbic acid synthesis and hepatic cytochrome P-450-linked monooxygenase systems by the interferon inducers, poly rI.rC and tilorone.

The interferon inducing agents, poly rI·rC and tilorone, cause a marked depression of hepatic cytochrome P-450-linked monooxygenase systems. Ascorbate synthesis and hepatic monnoxygenase systems are induced by phenobarbital and 3-methylcholanthrene. Poly rI·rC and tilorone suppressed the induction of ascorbate synthesis, P-450 and monooxygenase activity (ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase) by phenobarbital. 3-Methylcholanthrene-induced ascorbate synthesis was suppressed by poly rI·rC, but equivocal results were obtained with tilorone. Induction of P-450 by 3-methylcholanthrene was suppressed completely by poly rI·rC or tilorone, but that of benzo[a]pyrene hydroxylase was lowered by only 40%, thus demonstrating the selective depressive action of interferon inducing agents on different species of P-450.     

Differential control of rat microsomal "aryl hydrocarbon" monooxygenase and epoxide hydratase.

A growing body of evidence implicates epoxide metabolites of mutagenic and carcinogenic polycyclic hydrocarbons as either the only species, or one of the contributing species responsible for these adverse effects. Selective induction of epoxide hydratase(s) catalyzing the transformation of epoxides to electrophilically unreactive dihydrodiols, under conditions not leading to increases in monooxygenase(s) responsible for epoxide formation would, therefore, be of interest. All inducers of rat hepatic epoxide hydratase (determined with [7-3H]styrene oxide as substrate) which have been discovered also induced monooxygenase (determined with benzo(a)pyrene as substrate) suggesting a possible common biosynthetic control of these enzymes. The enzyme levels observed in different sexes and at different stages of the ontogenetic development, possibly dependent on endogenous inducers, strengthened this view. No sex difference is epoxide hydratase activity was observed in young rats (1 to 5 days old) while epoxide hydratase levels were about 3-fold higher in adult males than in females, which was remarkably similar to the behavior of monooxygenase. Moreover, the prenatal development of epoxide hydratase and monooxygenase appeared to be similar--although the low enzyme levels precluded accurate determinations of the latter. Although different types of known monooxygenase inducers all led to epoxide hydratase induction in adult rat liver, their effect of epoxide hydratase and monooxygenase could be dissociated by transplacental treatment. Dissociation was clearest with inducers of the polycyclic hydrocarbon type which led to great induction of monooxygenase while epoxide hydratase remained unchanged. The increases in monooxygenase activity were very different when determined by two methods based on different principles, demonstrating that at least two monooxygenases are involved in oxidative metabolism of benzo(a)pyrene, and that the control of epoxide hydratase is not under common control with either of them.     

Effects of non-ortho-substituted polychlorinated biphenyls (congeners 77 and 126) on cytochrome p4501a and conjugation activities in rainbow trout (Oncorhynchus mykiss)

Immature rainbow trout (Oncorhynchus mykiss) in two separate experiments received a single intraperitoneal injection of 0.1, 1 and 5 mg/kg of either 3,3′,4,4′-tetra- or 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC congeners 77 and 126, respectively). The experiments were run at water temperatures of 6 °C and 4 °C. Fish were killed 6 days after the injection. Biotransformation enzyme activities and cytochrome P4501A (CYP1A) amount and occurrence in different tissues were assayed. Congeners 77 and 126 strongly induced 7-ethoxyresorufin O-deethylase (EROD) and benzo(α)pyrene hydroxylase (AHH) activities in liver and kidney of rainbow trout. The induction of these cytochrome P4501A dependent monooxygenases was dose-related especially with congener 77 in the kidney. However, in the liver the highest dose of both congeners and in kidney the highest dose of congener 126 did not increase the catalytic monooxygenase activities as much as would have been expected based on the responses obtained with the lower doses. This may be because the monooxygenase activities already had attained their maximal induction capacity at 1 mg/kg dose of each congener. The PCB residues in liver were also determined and found to be highest after 5 mg/kg injections (610 μg/kg wet weight with congener 77 and 220 μg/kg with congener 126). When cytochrome P4501A protein content was measured, the induction of cytochrome P4501A was still on the increase even in those cases where catalytic activity failed to show any further induction. Immunohistochemical samples from liver, kidney and intestine showed cytochrome P4501A staining which strongly correlated with cytochrome P4501A in microsomes. Such observations suggest that the amount and occurrence of P4501A in the tissues can express the induction even when catalytic activities seem to be suppressed. With respect to enzymes mediating conjugation reactions, hepatic and renal UDP-glucuronosyltransferase (UDP-GT) activities showed elevated levels especially with the 1 and 5 mg/kg doses of both congeners. Glutathione S-transferase (GST) activities did not show such a clear trend. Congeners 77 and 126 preferentially affected the P4501A enzymes but to some extent also conjugation activities.     

Effect of nonsteroidal anti-inflammatory drugs on the microsomal monooxygenase system of rat liver

The effect of acetylsalicylic acid, ibuprofen, indomethacin, ketoprofen, naproxen, phenylbutazone, and salicylic acid on the microsomal oxidative drug metabolism of rat liver was studied. Pretreatment of the rats with pharmacologic doses of acetylsalicylic acid, indomethacin, and ketoprofen decreased both the demethylase and hydroxylase activities of rat liver microsomes. These effects were paralleled by decreases in microsomal cytochrome P-450 content. The rate of the microsomal reactions was increased after pretreatment with ibuprofen and naproxen but only the former increased the concentration of cytochrome P-450. Phenylbutazone and salicylic acid had no in vivo effect on the hepatic monooxygenase. The addition of 1 mM of ibuprofen, indomethacin, ketoprofen, naproxen, and phenylbutazone to rat liver microsomes inhibit both the aminopyrine N-demethylase and p-nitro-anisole O-demethylase activities. The extent of the inhibition varied between 21 and 73% of the control incubation. Indomethacin, naproxen, and phenylbutazone also decreased the aniline hydroxylase activity to roughly 60% of the control value. Acetylsalicylic acid and salicylic acid had no in vitro effect on the microsomal monooxygenase. The nonsteroidal anti-inflammatory drugs produced a reverse type I binding spectrum with oxidized cytochrome P-450; indomethacin and phenylbutazone were the strongest ligands. There is no correlation between the effect of addition of nonsteroidal anti-inflammatory drugs to the hepatic microsomal homogenate and their in vivo effect on the monooxygenase activity.     

Induction of cytochrome P-450 in congenic C57BL/6J mice by isosafrole: lack of correlation with the Ah locus

Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [B6(Ahd)]. Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 +/- 3.8 fmol/mg protein was measured in the hepatic cytosol from B6(Ahb) mice, while no receptor could be detected in the cytosol from B6(Ahd) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine N-demethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P2-450. In the case of 7-ethoxycoumarin O-deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6(Ahd) mice produced an equivalent or slightly larger induction of cytochrome P-450 than the B6(Ahb) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole.     

Cytochrome P450 induction by phenobarbital (PB) is inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA): evidence that protein kinase C regulates induction

The hepatic microsomal monooxygenase system was studied in hypophysectomized male rats exposed for 24 or 48 h to PB and/or TPA, an activator of kinase C. TPA attenuated basal and PB-induced levels of P450, aniline hydroxylase (ANH), ethylmorphine demethylase (EDM) and cytochrome c reductase. Hence, PB may effect induction via the inhibition of kinase C. Supporting this is spectral evidence that PB and TPA do not bind and the fact that TPA did not decrease P450 when co-incubated with O2 and NADPH. Hemin failed to increase P450 levels previously depressed by TPA indicating that TPA acts by lowering apocytochrome levels. This is consistent with its attenuation of PB-effected increases in hepatic RNA. TPA effects were associated with increased hepatic RNA and were blocked by puromycin.     

The effect of hepatic microsomal monooxygenase induction on the metabolism and toxicity of the organophosphorus insecticide chlorfenvinphos

The induction of rat liver microsomal monooxygenase by pretreatment of rats with dieldrin affords a 10-fold protection against the acute toxic effects of the organophosphorus insecticide, chlorfenvinphos. Metabolism studies were carried out to confirm that the protection was due to an enhanced rate of detoxification (via oxidative deethylation). At low doses of chlorfenvinphos (2.5 mg · kg^?1), dieldrin pretreatment caused minimal changes in the metabolic profiles. However, at a higher dose (13.2 mg · kg^?1), giving clinical signs of intoxication in the control animals, the dieldrin pretreated rats produced 5 times more deethylchlorofenvinphos than did the control animals. The results support the conclusion that the effect of enzyme induction on the metabolism of substrates of that enzyme are dosedependent. Alterations in metabolism, therefore, are not an automatic consequence of enzyme induction.     

Inducible Monooxygenase Activities and 3-Methylcholanthrene-Initiated Tumorigenesis in Mouse Recombinant Inbred Sublines

The induction of a certain group of hepatic monooxygenase activities by polycyclic aromatic compounds is regulated by the same locus or gene cluster controlling the formation of cytochrome P₁–450 (P–448) in mice. Certain inbred strains of mice are "responsive" (Ah^b) to such induction, whereas others are "nonresponsive" (Ah^d). A pair of closely related sublines that differ with respect to the Ah locus (for aromatic hydrocarbon responsiveness) were used to identify or confirm the pleiotropic effects of this gene. The lines were derived by sibling-mating without selection from (C57L/J x AKR/J)F ₂ mice; the two sublines were separated at the F₁₂ generation. Ten microsomal monooxygenase activities and one cytosol enzyme activity known to be associated with the Ah locus were similarly associated with cytochrome P₁–450 formation in these recombinant inbred sublines as well. Nine additional hepatic monooxygenase activities studied were found not to be associated with the Ah locus; certain of these activities were increased slightly, following treatment of nonresponsive as well as responsive mice with polycyclic aromatic compounds. The Ah^b-containing subline was highly susceptible to 3-methylcholanthrene-induced subcutaneous sarcomas, whereas the Ah-^d-containing subline was relatively resistant. These results emphasize the potential importance of this particular enzyme for the study of coordinated regulation in mammals.     

Activation of polycyclic hydrocarbons in Reuber H4-II-E hepatoma cells an in vitro system for the induction of SCEs

Activation of polycyclic aromatic hydrocarbons (PAHs) was examined in the Reuber H4-II-E established cell line without the use of exogenous enzyme preparations. Metabolism of PAHs to genotoxic products was determined by the induction of sister-chromatid exchanges (SCEs). The induction of SCEs followed a dose-response pattern with plateaus at high doses of PAH. The effects of metabolic enzyme inducers (3-methylcholanthrene, phenobarbital, Aroclor 1254) and the epoxide hydrase inhibitor 1,1,1-trichloropropylene oxide were assessed as changes in SCE induction and enhanced production of water-soluble metabolites. Results indicate that Reuber H4-II-E cells can be employed in the testing of carcinogens activated by the P₁-450 monooxygenase system and would be a useful in vitro system for the study of mechanisms of metabolic induction and their effect on genetic toxicity.     

Decreased placental monooxygenase activities associated with birth defects

We have measured monooxygenase activities in placentas from 82 women who smoked throughout their pregnancies and correlated these with the presence or absence of major somatic anomalies. Monooxygenase activities toward benzo(a)pyrene and ethoxyresorufin in placentas from 18 abnormal infants were compared with activities in placentas from 64 concurrently studied normal infants. Placentas from normal infants were found to have high levels of monooxygenase activities and low apparent Kms toward ethoxyresorufin (10(-7) M), reflecting induction of cytochrome P-450 enzymes usually associated with maternal cigarette smoking. Placentas from the abnormal infants, however, had significantly lower monooxygenase activities and higher apparent Kms toward ethoxyresorufin (10(-5) M), indicating that induction of specific cytochrome P-450 systems occurred less frequently among placentas from abnormal infants. The reasons for this association are unclear. Apparent lack of induction of monooxygenase activity occurred most frequently in placentas from anencephalic infants but was neither exclusively nor consistently found with this defect. No specific maternal condition or environmental exposure associated with lack of monooxygenase induction was identified.     

Heterogenous effects of anthraquinones on drug-metabolizing enzymes in the liver and small intestine of rat

The induction of a variety of drug-metabolizing enzymes by six anthraquinones (AQs) has been investigated in the liver and small intestine of rat. In the liver, the intragastric administration for 3 days of 100 mg/kg 9,10-anthraquinone (9,10-AQ). 1-hydroxy-AQ, 1,4-dihydroxy-AQ, but not 1,2-dihydroxy-AQ and 2-carboxy-AQ, resulted in a significant induction of the UDP-GT, DT-diaphorase, P450 1A-linked monooxygenase activities and in particular the methoxyresorufin-O-demethylase (MEROD), an activity dependent on P450 1A2. Immunoblot analysis indicated that 1-hydroxy-AQ and 1,4-dihydroxy-AQ induced P450 1A2 but not 1A1 and 9,10-AQ induced both P4501A2 and P4502B. Northern blotanalysis, using a cDNA probe for CYP 1A1 and CYP 1A2, confirmed that the AQs induce CYP 1A2 but not 1A1 mRNA. In the mucosa of small intestine, none of the above-mentioned enzymatic activities were enhanced following AQ administration. The induction mechanism of the hepatic enzymes by AQs is not known and it deserves a further study as it might be independent from the activation of the Ah-receptor as reported for other tricyclic compounds. The results from inhibition experiments showed that the hydroxylated AQs were strong inhibitors of P450 1A2-dependent monooxygenases. This suggests that long-term ingestion of certain AQs, may affect the toxicity of other components present in the diet through the hepatic induction or inhibition of P450 1A2.     

A study of the hepatic microsomal monooxygenase of sea birds and its relationship to organochlorine pollutants

1. The levels of hepatic microsomal monooxygenase in sea birds were determined using organochlorine substrates. Levels of cytochrome P450 and organochlorine residues were also measured. 2. The razorbill (Alca torda) and puffin (Fratercula arctica) showed highly variable activities which were resolved into multiple peaks on frequency diagrams. 3. The most active individuals amongst razorbills were early season females with large ovaries. 4. The properties of monooxygenase from individuals of low and high activity were compared. 5. The results are discussed in relation to PCB pollution.     

Tissue specificity of induction of hamster monooxygenase activity by ethanol

The effects of ethanol on liver, kidney and intestine monooxygenases were studied using hamsters chronically fed with isocaloric control and ethanol-containing liquid diets. The inductive effects of ethanol on liver and kidney aniline hydroxylase activities began to approach plateau level after the animals were fed ethanol for two weeks. Intestinal aniline hydroxylation was refractory to ethanol induction. In control and ethanol-fed hamsters, CO-difference spectra of hepatic and extrahepatic microsomes differed in absorption maxima. Chronic alcohol consumption caused significant increases of cytochrome P-450 and cytochrome b5 contents of liver and kidney microsomes. The increases of the heme proteins were associated with the induction of aniline hydroxylase, N-nitrosodimethylamine demethylase and 7-ethoxycoumarin 0-deethylase activities. In contrast to the liver and kidney, intestinal microsomal cytochromes P-450 and b5 contents in ethanol-treated animals were lower than the controls. Ethanol pretreatment was without effect on intestinal monooxygenase activities toward the metabolism of aniline, N-nitrosodimethylamine, 7-ethoxycoumarin and benzo(a)pyrene. Gel electrophoresis of tissue microsomes from control and ethanol-treated hamsters revealed that ethanol treatment enhanced the intensity of the protein band(s) in the cytochrome P-450 molecular weight region in the liver and kidney, but not in the intestine. These results demonstrate that in hamsters the response of monooxygenase to ethanol may vary from tissue to tissue and it is difficult to make a generalization regarding the inducing property of ethanol. The differential effect on cytochrome P-450 may be an important factor in determining the interaction between ethanol and xenobiotic metabolism in animal tissues.     

The effect of monooxygenase inducing agents on the incorporation of [35S]methionine into hepatic microsomal protein of rainbow trout (Salmo gairdneri)

Treatment of rainbow trout (Salmo gairdneri) with 150 mg/kg BNF resulted in an increase in hepatic microsomal monooxygenase activity as assessed by ECOD and EROD when compared to those activities in corn oil-pretreated animals. Administration of 100 mg/kg 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) to trout had no significant effect on these catalytic activities or on BeND. The amount of radioactivity in hepatic microsomes at 24, 48 or 72 hr following the administration of 75 muCi of [35S]methionine was consistently higher in animals pretreated with BNF than in those treated with corn oil or 6-CB. Autoradiography/fluorography of electrophoretograms demonstrated the appearance of at least three radiolabeled bands in the 50,000-60,000 mol. wt range in solubilized microsomes from BNF-treated fish which were not present in microsomes from control animals or fish treated with 6-CB. These data indicate that the stimulation of hepatic microsomal catalytic activities observed following the administration of 3-MC-type agents to rainbow trout is due, at least in part, to induction of enzyme(s) rather than activation of existing enzyme(s). These results further support the observation that fish appear to be non-responsive to phenobarbital-type inducing agents.     

2,3,7,8-Tetrachlorodibenzo-P-dioxin: potent anticarcinogenic activity in CD-1 mice.

Topical pretreatment with non-toxic doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a contaminant formed in the commercial synthesis of the herbicide 2,4,5-trichlorophen-oxyacetic acid, strongly inhibited the initiation of skin tumors by 7,12-dimethylbenz(a)-anthracene and benzo(a)pyrene in female CD-1 mice. 2,3,7,8-tetrachlorodibenzo-p-dioxin also produced marked induction of epidermal monooxygenase enzymes functional in the conversion of 7,12-dimethylbenz(a)anthracene to a variety of hydroxylated products. The time course of anticarcinogenic effects resulting from pretreatment with the dioxin correlated with the magnitude of induction as well as with a singnificant reduction in the quantity of 7,12-dimethylbenz(a)anthracene metabolites covalently bound $\text{in vivo}$ to epidermal DNA and RNA but not protein.