Variation in aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity in heteroploid and predominantly diploid rat liver cells in culture.

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase is present and inducible in Buffalo rat liver cells in culture. There is substantial variation in both basal and inducible hydroxylase activities among heteroploid subclones isolated from a heteroploid parent population, and among diploid subclones isolated from a diploid parent population. This variation is not related to differences in the growth characteristics of the subclones, or to differences in their chromosome number. The results indicate that substantial heterogeneity in both basal and induced hydroxylase activity develops during the growth of both heteroploid and diploid cell strains in culture. These findings indicate that diploid cell populations are not necessarily homogeneous with respect to aryl hydrocarbon hydroxylas activity. This observation may complicate the interpretation of experiments involving somatic cell hybridization or polycyclic hydrocarbon-induced transformation and/or cytotoxicity. This heterogeneity in hydroxylase activity develops rather rapidly (2-3 mo of culture), in the absence of any apparent mutational stress.

Field evaluation of benzopyrene hydroxylase induction as a monitor for marine petroleum pollution.

Fish from petroleum-contaminated sites in the marine environment have elevated levels of benzopyrene hydroxylase activity in liver and gill tissue. This sublethal response appears to be a practical biological monitor for marine petroleum pollution.

Metabolism of benzo(a)pyrene by guinea pig pancreatic microsomes.

The in vitro microsomal metabolism of the strain 13 guinea pig pancreas was investigated by determining the benzo(a)pyrene (BP) hydroxylase activity in the 9000 x g supernatant and microsomal pellet. BP hydroxylase activity in both 9000 x g supernatant and microsomal pellet of the pancreas was less than 1% of the activity in the respective liver fractions, However, pretreatment of animals with methylcholanthrene or BP at 20 mg/kg, for either 1 day or 3 consecutive days, markedly enhanced the BP hydroxylase activity of pancreatic microsomes over that of controls; the induction in the liver microsomes was less than 2-fold over that of controls. The hydroxylation of BP by pancreatic microsomes was linear with time over a 30-min period, with the rate of hydroxylation dependent on both the enzyme and substrate concentrations.

Reappraisal of the liver benzpyrene hydroxylase synthesized de novo after treatment of rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene.

The dynamics of the inductive effects of MC and TCDD upon rat liver microsomal benzpyrene hydroxylase and the main properties of the de novo synthesized hemoproteins have been compared. The inadequacy of expression of the enzyme activity per total cytochrome P-448 content has been established. It was concluded that TCDD microsomes have a relatively low content of benzpyrene hydroxylase with a higher molecular activity than the enzyme from MC microsomes.

Localization and induction of cytochrome P450 1A1 and aryl hydrocarbon hydroxylase activity in rat nasal mucosa.

Cytochrome P450 1A1 was localized immunohistochemically and benzo[a]pyrene hydroxylase activity was identified in situ by means of fluorescence histochemistry in the nasal mucosa of untreated, 3-methylcholanthrene-treated or Aroclor 1254-treated rats. Cytochrome P450 1A1 was localized predominantly within Bowman's glands, with considerably less staining occurring in the olfactory epithelium of untreated rats. Similarly, benzo[a]pyrene was hydroxylated to the greatest extent in Bowman's glands and, to a lesser extent, in olfactory epithelial cells. Pre-treatment of tissue sections of nasal mucosa with anti-P450 1A1 inhibited most of the benzo[a]pyrene hydroxylase activity present. Although 3-methylcholanthrene treatment did not affect either cytochrome P450 1A1 or hydroxylase activity in the nasal mucosa, a single intraperitoneal injection of Aroclor 1254 significantly increased anti-P450 1A1 binding in Bowman's glands and in the olfactory and respiratory epithelia, and dramatically enhanced benzo[a]pyrene hydroxylase activity in the epithelia and the subepithelial ducts and glands in both the olfactory and respiratory regions. In contrast to its effects on cytochrome P450 1A1, Aroclor 1254 produced a considerably greater induction of hydroxylase activity in the respiratory region, especially in the seromucous glands, than in the olfactory region. These results suggest that Aroclor 1254 treatment also induces other forms of cytochrome P450 in the respiratory region of the nasal mucosa.

Polycyclic aromatic hydrocarbon profiles of pyrolysed tobacco products commonly used in India.

The polycyclic aromatic hydrocarbon (PAH) profile of bidi (an indigenous substitute for cigarettes in India), masheri (a charred tobacco product used for cleaning teeth) and Indian snuff was determined. The effect of the extracts of these tobacco products on the liver microsomal mixed function oxygenase system was studied. Cytochrome P-450 and benzo[a]pyrene (B[a]P) hydroxylase levels were significantly increased.

Characterization of the microsomal cytochrome P-450 species induced in rat liver by trans-stilbene oxide.

trans-Stilbene oxide differs from the classical inducers of drug-metabolizing enzymes, phenobarbital and 3-methylcholanthrene, in that it induces the so-called phase II activities, epoxide hydrolase and glutathione S-transferase, to a much larger extent than it induces cytochrome P-450. Nonetheless, the level of cytochrome P-450 in liver microsomes from rats treated with trans-stilbene oxide is increased significantly to twice the control value. The existence of a number of different isozymes of cytochrome P-450 has now been clearly demonstrated and in the present study we have posed the question. What form(s) of cytochrome P-450 is induced by trans-stilbene oxide? A number of criteria including substrate specificity, pattern of benzo(a)pyrene metabolism, sensitivity to inhibitors, substrate binding spectra, ethylisocyanide binding spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and crossed immunoelectrophoresis were used to answer this question. It seems clear that trans-stilbene oxide induces the same form(s) of cytochrome P-450 as phenobarbital.

Differential induction of rat liver microsomal UDP-glucuronosyltransferase activites by various inducing agents.

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methyl-cholanthrene beta-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT1 substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT2 substrates) and bilirubin, similar to phenobarbital. (3) Colfibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT1 and GT2 reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT1 and GT2 activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450MC and GT1, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced GT1 reactions without affecting benzo[a]pyrene monooxygenase.

Relationship between the antiviral effects of interferons and their abilities to depress cytochrome P-450.

Several hybrid human interferons have now been constructed by recombinant DNA techniques. Two of these hybrid interferons, IFN-alpha AD(Bgl) and IFN-alpha AD(Pvu) differ by only three amino acids, but IFN-alpha AD(Bgl) was fifteen times more potent than IFN-alpha AD(Pvu) in antiviral activity towards infection of mouse L-929 cells by vesicular stomatitis virus. Only the hybrid with the greater antiviral activity in the mouse depressed cytochrome P-450, aminopyrine N-demethylase and benzo[a]pyrene hydroxylase in the liver. These experiments demonstrate that minor changes in amino acid structure not only have a major effect on the antiviral properties of interferon but also influence the ability of interferon to depress cytochrome P-450 in the liver.

Localization and characterization of drug-metabolizing enzymes along the villus-crypt surface of the rat small intestine--I. Monooxygenases.

To investigate the drug-metabolizing potential of different sub-populations of cells along the villus-crypt surface of the small intestine, the major monooxygenase activities directed towards the substrates benzo[a]pyrene (BP), 7-ethoxycoumarin and ethylmorphine were studied. The cells were isolated in sequential fractions corresponding to the villus tip-to-crypt gradient in the small intestinal epithelium of the rat. Cells from the upper- and mid-villus regions were rich in aryl hydrocarbon (BP)hydroxylase (AHH) and 7-ethoxycoumarin deethylase (7-ECDE) activities whereas in crypt cells the activities of these enzymes were at the level of detectability. Ethylmorphine demethylase (EMD) was not detectable in the entire villus-crypt surface. The intestinal epithelial cells responded strongly to inducers. 3-Methylcholanthrene (3-MC), given to rats 24 hr previously, induced increases in AHH activity of 4- to 7-fold in the villus and of 19- to 26-fold in the crypt cells. 7-ECDE had a similar pattern. The induced level of monooxygenase activity in crypt cells was sustained for a longer time, followed in order by consecutively higher cells of the villus. Phenobarbital caused maximal expression of EMD activity in the mid-villus region whereas the activity in crypt cells was half the maximal activity. PB also significantly induced AHH and 7-ECDE in the intestinal epithelium. 7,8-Benzoflavone inhibited AHH activity to the same degree in all the cell fractions. The apparent Km for AHH was 5 microM (BP). Treatment of rats with 3-MC, after 24 hr, enhanced the Km and Vmax differently in the cells along the villus-crypt surface. The Km value in the villus region increased, whereas it did not change in the crypt cells; Vmax increased 6-fold in the villus and about 12-fold in the crypt cells, above their basal levels. The results suggest that the intestinal cells are capable of biotransforming various xenobiotics. The higher sensitivity of their monooxygenases, particularly of the crypt cells, might protect them directly or render the cells capable of generating metabolites that aid and abet toxicity toward target tissue in vivo.

Dietary fat--a requirement for induction of mixed-function oxidase activities in starved-refed rats.

Male rats were starved 0-48 hr, and then refed diets containing 0% (F.F.) to 20% corn oil (C.O.) lab chow or 20% coconut oil (C.C.O.) for 1-4 days. Some received phenobarbital sodium (80 mg/kg, i.p. daily) for 1-3 days prior to decapitation. Five cytochrome P-450-dependent indicators were assayed as measures of altered hepatic microsomal function: ethylmorphine N-demethylase (EMDM), N-nitrosodimethylamine (DMN)-N demethylase, aniline hydroxylase (AH), benzo[a]pyrene hydroxylase (AHH) and CO-difference spectra (P-450). Increasing dietary corn oil (0, 0.5, 10, 20%) in control rats resulted in a progressive increase in the activities of these five enzymes. Dietary fat influenced phenobarbital (Pb) inducibility of all mixed-function oxidase (MFO) enzymes measured except AHH. Pb induced the remaining enzymes only 11-22% in animals fed fat-free diet as compared to 119-246% in animals fed coconut oil and corn oil. Rats fed fat-free diet for 21 days without prior food deprivation and administered Pb had 79% more EMDM, 34% more AH and 120% more P-450 than non-induced controls, whereas rats fed 20% corn oil diet had 227% more EMDM, 143% more AH and 128% more P-450. A requirement of dietary fat for induction of MFO by Pb was demonstrated by these starvation-refeeding experiments. Coupled with data recovered from the 21-day studies, these experiments suggest that a compensatory mechanism may be operative during chronic feeding of the fat-free diet to partially return inducibility to the drug-metabolizing system.

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice.

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.

Induction of cytochrome P-450 and related drug-metabolizing activities in the livers of different rodent species by 2-acetylaminofluorene or by 3-methylcholanthrene.

In general, large differences in the control levels of different cytochrome P-450-catalyzed activities (aminopyrine N-demethylase, benzo(a)pyrene monooxygenase, ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase and total 2-acetylaminofluorene metabolism and metabolite pattern) and in the inducibility of these activities in different rodent species (rat, hamster, guinea pig and mouse) and sexes were observed. For all the activities measured the lowest levels were observed in untreated rats. With a few minor exceptions, the only species tested in which cytochrome P-450-catalyzed activities were induced by treatment with 2-acetylaminofluorene was the rat. A larger number of the species tested were susceptible to induction by 3-methylcholanthrene. However, this xenobiotic proved also to induce most potently in the rat. There are relatively large differences between the male and female rat both in terms of control cytochrome P-450-catalyzed activities and in the inducibility of these activities by 2-acetylaminofluorene and 3-methylcholanthrene. In general, both of these xenobiotics proved to be more potent inducers in the female than in the male. Thus, it is quite clear that in quantitative terms the hepatic microsomal cytochrome P-450-catalyzed activities and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene in the male Sprague-Dawley rat are not representative for other rodent species or even for the female of the same species.

Selective induction of two different molecular species of cytochrome P-450 by phenobarbital and 3-methylcholanthrene.

Two forms of cytochrome P-450, P-450PB and P-450MC, were purified to homogeneity from the liver microsomes of phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated rats, respectively. Rabbit antibodies against P-450PB and P-450MC were prepared and the monospecificity of each antibody preparation was confirmed by various lines of evidence. By the use of these antibodies, the contents of P-450PB and P-450MC in microsomes could be determined separately by quantitative immunoprecipitation. P-450PB and P-450MC each amounted to about 10% of total cytochrome P-450 in the microsomes of normal rats. However, each of them was selectively and substantially increased by the appropriate inducer, and became a predominant component of cytochrome P-450 in the microsomes of drug-treated animals. The increase of each specific molecular species of cytochrome P-450 was about 10- and 20-fold within 48 h, whereas the increase of total cytochrome P-450 was only about 2- to 3-fold even after maximal induction by the drugs. The drug oxidation activities of P-450PB and P-450MC were also significantly altered by the drug treatments. The administration of MC to PB-treated rats induced a drastic decrease in the P-450PB-dependent oxidations of benzo(a)pyrene and 7-ethoxycoumarin, while the corresponding activities of P-450MC increased sharply, and these changes were much more rapid than the change of the amount of each form of cytochrome P-450. However, the oxidation of benzphetamine was almost exclusively P-450PB-dependent even after extensive induction by MC. These observations suggest that the specific activities of p-450PB and P-450MC in the oxidations of various drugs are quite differently affected by the induced states of animals.

The biologic and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in chickens.

The administration of 2,3,7,8-TCDD to 2-week-old White Leghorn cockerels produced a dose-dependent induction of hepatic microsomal ethoxyresorufin O-deethylase (EROD) and benzo[alpha]pyrene (B[alpha]P) hydroxylase activities with induction EC50 values of 778 and 302 ng/kg, respectively. In addition, 2,3,7,8-TCDD also induced 4-dimethylaminoantipyrine (DMAP) N-demethylase (EC50 = 561 ng/kg), this result contrasting with studies reported for other animal species in which 2,3,7,8-TCDD either does not induce or inhibits this cytochrome P-450 dependent monooxygenase enzyme activity. However, the reduced cytochrome P-450:CO binding difference spectral absorption maxima for the 2,3,7,8-TCDD induced microsomes was observed at 448 nm which was similar to that reported for most animals which have been investigated. Electrophoresis of control and 2,3,7,8-TCDD induced microsomal proteins using SDS polyacrylamide slab gels showed intensification of 3 protein staining bands at Mr 53 000, 56 000 and 58 000. Incubation of the 2,3,7,8-TCDD-induced microsomes with SKF-525A and alpha-naphthoflavone showed that both compounds inhibited DMAP N-demethylase, EROD and AHH and that alpha-naphthoflavone was the more potent inhibitor of all 3 microsomal monooxygenases. 2,3,7,8-TCDD treatment caused significant involution of the Bursa of Fabricius when administered at a dose level of 10 micrograms/kg for 3 days and this result confirmed the extreme sensitivity of the immature White Leghorn cockerel to the biologic and toxic effects elicited by 2,3,7,8-TCDD. However, in contrast to other sensitive species, no high affinity cytosolic receptor protein for [3H]2,3,7,8-TCDD could be detected in the liver of chick embryos or 2-week-old birds.

Induction of benzo[a]pyrene hydroxylase in skin and liver by cutaneous application of jute batching oil.

Skin painting with 30 microliter of jute batching oil (JBO) for 8 days resulted in increased gross liver weight, microsomal protein content and benzo[alpha]pyrene hydroxylase activity of liver. Skin benzo[alpha]pyrene hydroxylase activity at the treated site increased by 10-fold. An investigation of cytochrome pigment status in liver and skin of treated animals showed a specific increase in P-448 level in both tissues. Single skin applications of JBO elevated the level of skin and liver benzo[alpha]pyrene hydroxylase activity to its maximum after 1 day and 2 days, respectively, which, in absence of further treatment with mineral oil, declined gradually to normal levels in due course. The results suggest that single or multiple cutaneous exposure(s) with JBO can increase carcinogen metabolising status of skin and liver which may be one of the causative factors for the tumorigenic effects of JBO in skin.

PCBs and PBBs: biologic and toxic effects on C57BL/6J and DBA/2J inbred mice.

Treatment of genetically inbred "responsive" C57BL/6J and "non-responsive" DBA/2J mice with Aroclor 1254 or fireMaster BP-6 resulted in the induction of hepatic microsomal benzo[a]pyrene hydroxylase only in the former mouse strain and aminopyrine N-demethylase in both strains of mice. In contrast, 3,3',4,4',5-pentachlorobiphenyl and 3,3',4,4'-tetrabromobiphenyl, induced benzo[a]pyrene hydroxylase in both C57BL/6J and DBA/2J but did not enhance aminopyrine N-demethylase in either strain of mouse. Both these coplanar halogenated biphenyls also caused thymic atrophy in the responsive and non-responsive mice and their effects resembled those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Treatment of the inbred mice with several mono-ortho substituted analogs of the coplanar halogenated biphenyls, including 2,3,3',4,4'-pentachloro-, 2,3',4,4',5-pentabromo-, 2,3,3',4,4',5-hexachloro- and 3',4'-dibromo-2,3,4,5-tetrachlorobiphenyl, gave hepatic enzyme-induction results similar to those observed for the commercial halogenated biphenyls. At dose levels of 1500 mumol/kg, most of these compounds caused thymic atrophy in C57BL/6J mice but not in DBA/2J mice. The structure-activity correlations in the mice complement similar studies with the halogenated biphenyls in rats and support the proposed receptor-mediated mechanism for the toxic halogenated aromatics.

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on cytochrome P-450-dependent monooxygenase activities in rat hepatoma cells: induction of P-450I and suppression of P-450II.

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytochrome P-450-dependent monooxygenase activities in several differentiated and dedifferentiated Reuber rat hepatoma cell lines using aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and aldrin epoxidase (AE) as test systems. The following results were obtained: (1) Exposure of cultures to 400 nM TPA for 18-24 h increased AHH activities in the differentiated lines 2sFou, H41IEC3/G- and Fao as well as in the dedifferentiated line 5L, 1.5-2.5-fold. The phorbol ester did not affect AHH activity in the dedifferentiated line H5. (2) EROD, a marker for P-450I, was induced by the phorbol ester to a similar degree as AHH. (3) A monoclonal antibody directed against P-450I strongly inhibited the AHH activity induced by TPA. (4) The onset of AHH or EROD induction by TPA was much later than that elicited by benz[a]anthracene. (6) In contrast to the induction of AHH and EROD, TPA decreased AE activity, a marker for P-450II, by about 50% in all the cell lines containing this monooxygenase activity. (7) The half-maximum-effect concentration of TPA for inducing or suppressing AHH and AE, respectively, was approximately 20 nM. (8) TPA did not interfere with AHH induction by benz[a]anthracene. However, the phorbol ester moderately decreased AHH induction and markedly suppressed AE induction by dexamethasone. The results indicate that TPA simultaneously induces P-450I and suppresses P-450II forms in rat hepatoma cells. P-450I induction by TPA in these cells did not appear to depend on their status of differentiation. Furthermore, the results suggest that the mechanism of P-450I induction by TPA differs from that elicited by polycyclic aromatic hydrocarbons or glucocorticoids.

Biological activity of 2,4,8-trichlorodibenzofuran: promotion of rat liver foci and induction of cytochrome P-450-dependent monooxygenases.

The biological activity of 2,4,8-trichlorodibenzofuran (2,4,8-TCDF) was tested using 2 endpoints: (a) the promotion of enzyme-altered, preneoplastic foci initiated by diethylnitrosamine (DEN) in livers of weanling female Sprague-Dawley rats; and (b) the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), a marker for cytochrome P-4501 activity, in livers of adult female Sprague-Dawley rats and in H4IIEC3 rat hepatoma cells. When animals were treated with 200 or 500 mg/kg 2,4,8-TCDF 5 X weekly over 10 weeks after a single application of 10 mg/kg DNA, the higher dose of 2,4,8-TCDF had a promoting effect on the appearance of preneoplastic foci. Thus number and total area of foci deficient in adenosine-5'-triphosphatase were significantly increased by a factor of 1.6. 2,4,8-TCDF induced AHH-activities in 9000 X g supernatants of liver 2-3-fold, when rats were treated with 100-1000 mg/kg/day for 5 days and monooxygenase activities determined after another 3 days. The amounts of 2,4,8-TCDF required for inducing AHH activity in H4IIEC3 cells were 7 orders of magnitude higher than those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). the results indicate that the 2,4,8-TCDF has a biological activity which is extremely low compared to that of 2,3,7,8-TCDD.

Selective induction and inhibition of liver and lung cytochrome P-450-dependent monooxygenases by the PCBs mixture, Aroclor 1016.

The effects of the widely used industrial PCBs mixture, Aroclor 1016, as modifiers of monooxygenases were studied in rats and rabbits. From data presented, it is not possible to generalize the biological effects of PCBs observed with rats, namely, that they are potent, non-specific inducers of monooxygenase activities. In rat liver, Aroclor 1016 exhibited primarily the potent inducing effects of the barbiturate class of inducers. In contrast, in rabbits pretreated with Aroclor 1016, although cytochrome P-450 content of the liver was significantly increased, benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities were decreased 30-35%; and no changes in the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin were observed. These results strongly suggest differences in the regulation of cytochrome P-450 isozymes in the liver by the PCBs. A similar conclusion can be drawn from the pulmonary studies of Aroclor 1016-pretreated rabbits. In the lung, cytochrome P-450, form 2 and associated enzymic activities were markedly decreased, with little or no effect on the form 5 isozyme. Electrophoretic and chromatographic studies confirmed these findings. The induction and the repression of a form or forms of cytochrome P-450 by environmentally-derived chemicals may be important determinants of organ-targeted chemical toxicity.