Characterization of the hepatic and splenic immune status and immunoglobulin synthesis in aged male mice lacking the peroxisome proliferator-activated receptor-alpha (PPARα).

It is now well established that the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARα) is expressed in different types of immune cells and plays a pivotal role in the regulation of age-related production of inflammatory cytokines. However, the role(s) of this receptor in the regulation of immune cell homoeostasis in ageing non-lymphoid and lymphoid organs has not yet been resolved. We examine this issue here by evaluating the hepatic and splenic immune status and immunoglobulin (Ig) production in male PPARα-null mice and their wild-type littermates at one and 2 years of age. In comparison with the age-matched control animals, PPARα-null mice exhibited age-related elevations in the numbers of total, as well as of phenotypically distinct subpopulations of intrahepatic immune cells (IHIC) and splenocytes. Moreover, at 2 years of age, these alterations in hepatic immune cells were accompanied by significant increases in hepatic levels of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interferon-gamma (IFN-γ), in combination with the development of hepatic inflammatory loci containing mixtures of leucocytes. Alterations in splenocytes of old PPARα-null mice were also accompanied by increases in cellularity of both white and red pulps of the spleen. Furthermore, these same animals exhibited pronounced increases in the numbers of splenic plasma cells and enhanced production of Ig of different isotypes, including IgG1, IgG2a and IgE. Thus, our findings indicate that upon ageing, PPARα plays a crucial role in regulating the total numbers, compositions and functions of immune cells in both lymphoid and non-lymphoid immune organs of mice.

Isolation of murine intrahepatic immune cells employing a modified procedure for mechanical disruption and functional characterization of the B, T and natural killer T cells obtained.

Intrahepatic immune cells (IHIC) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. In the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of IHIC. These cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and DNase. The mechanical disruption yielded viable IHIC in considerably greater numbers than those obtained following enzymatic digestion. The IHIC isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which B, T, natural killer (NK), NK T cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). The IHIC obtained following enzymatic digestion contained markedly lower numbers of NK T cells (1.8%). The B, T and NK T cells among IHIC isolated employing mechanical disruption were found to be immunocompetent, i.e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin A and alpha-galactosylceramide respectively) and produced immunoglobulin M and interferon-gamma. Thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent IHIC for various types of investigation.

Ecto-enzyme of granulocytes: 5'-nucleotidase.

The 5'-nucleotidase of guinea pig polymorphonuclear leukocytes is localized exclusively on the plasma membrane of intact cells. The active site of this enzyme faces the external medium, not the cytoplasm.

Antidepressant-induced lipidosis with special reference to tricyclic compounds.

Cationic amphiphilic drugs, in general, induce phospholipid disturbances. Tricyclic, as well as other antidepressants belong to this group. In experimental animals, antidepressants induce lipid storage disorders in cells of most organs, a so-called generalized phospholipidosis. This disorder is conveniently detected by electron microscopic examination revealing myelin figures. Myelin figures or myeloid bodies are subcellular organelles containing unicentric lamellar layers. The lipidotic induction potency during in vivo is related to the apolarity of the compound. Metabolism of phospholipids takes place within the cell continuously. Several underlying mechanisms may be responsible for the induction of the phospholipid disturbance. For instance, it has been suggested that the compounds bind to phospholipids and such binding may alter the phospholipid's suitability as a substrate for phospholipases. Free TCA or metabolites thereof may also inhibit phospholipases directly, as has been demonstrated for sphingomyelinase in glioma and neuroblastoma cells. Both these mechanisms might result in phospholipidosis. Interaction between drug and phospholipid bilayer has been investigated by nuclear magnetic resonance technique. There seems to be large differences in the sensitivities amongst different organs. Steroid-producing cells of the adrenal cortex, testis and ovaries are in particular susceptible to drug-induced lipidosis. The so-called foam cells are lung macrophages located in the interstitium which become densely packed with myelin figures during TCA exposure. It requires about 3-6 weeks of treatment to develop this converted cell. In cell cultures however, phospholipidosis is demonstrated already after 24 h only. It appears that the cells that undergo TCA-induced lipidosis may recover after withdrawal of the drug. The time required to achieve complete recovery ranges from 3-4 weeks to several months, depending on the organ affected. Little is known about the functional significance of lipidosis. Even if TCA and other antidepressants show other effects, it has not been possible to exclusively relate it to phospholipidosis. However, few attempts have been made to correlate the physiological effects of TCAs in experimental animals to the morphological changes associated with phospholipidosis. There is an increasing evidence however, that cationic amphiphilic drugs may have effects on immune function, signal transduction and receptor-mediated events, effects that to some extent might be related to disturbances in phospholipid metabolism.

Characterization of the microsomal cytochrome P-450 species induced in rat liver by 2-acetylaminofluorene.

2-Acetylaminofluorene induces the level of cytochrome P-450 in rat liver microsomes by 50% (p less than 0.001). This induced cytochrome(s) was characterized and compared to the major forms of cytochrome P-450 induced by phenobarbital and 3-methylcholanthrene. The properties investigated were: the absorption maximum of the complex formed between reduced cytochrome P-450 and carbon monoxide; the substrate specificities using aminopyrine, benzphetamine, ethylmorphine, benzo(a)pyrene, ethoxycoumarin, ethoxyresorufin, and 2-acetylaminofluorene itself as substrates; metabolite patterns with benzo(a)pyrene and 2-acetylaminofluorene; sensitivity to different inhibitors; binding spectra with aniline and hexobarbital; and molecular weight as determined by sodium dodecyl sulfate:disc gel electrophoresis. The results indicate that 2-acetylaminofluorene induces a form(s) of cytochrome P-450 especially effective in the metabolism of this substance itself (i.e., the process can be called substrate induction) and different from the major forms of cytochrome P-450 induced by phenobarbital and 3-methylcholanthrene.

Measurement and characterization of membrane-bound and soluble epoxide hydrolase activities in resting mononuclear leukocytes from human blood.

Membrane-bound and soluble epoxide hydrolase activities in the mononuclear cell fraction from human blood have been characterized using cis- and trans-stilbene oxides as substrates, respectively. Because of the low activities in these cells, it was necessary to modify assay procedures developed for rat and mouse liver in the following ways: (a) the substrates were relatively highly labeled (2 Ci/mmol) and carefully purified; (b) the incubation time was extended to 45 to 60 min, during which period the activities were linear; (c) as many as 6 million cells were used for a single assay, which was also within the linear range of the procedure. The membrane-bound epoxide hydrolase characterized in this manner has an apparent Vmax of 7.26 pmol product formed per min per 10(7) cells and an apparent Km of 9.96 microM. The pH optimum was observed to be around 9.8. The dependence of this activity on temperature showed its optimum at 40 degrees. The soluble epoxide hydrolase activity has an apparent Vmax of about 8.26 pmol product formed per min per 10(7) cells, an apparent Km of 1.63 microM, a pH optimum of 6.2 to 6.8, and a temperature optimum at 60 degrees. Using these techniques, these activities have also been determined in other blood components, i.e., lymphocytes, monocytes, granulocytes, erythrocytes, platelets, and plasma. Lymphocytes account for most of the epoxide hydrolase activity towards cis-stilbene oxide, and all of the activity towards trans-stilbene oxide is in the human mononuclear cell fractions. Different substances known to affect rodent epoxide hydrolases were tested for their effects on the human mononuclear blood cell activities. Interestingly, 1,1,1-trichloropropane 2,3-epoxide, a potent inhibitor of liver microsomal epoxide hydrolase in different species including rat, mouse, and human, had little or no effect on the membrane-bound activity measured here. However, cyclohexene oxide inhibits this membrane-bound activity 60%. The soluble epoxide hydrolase is inhibited to 90% of control levels by chalcone epoxide. The membrane-bound and soluble epoxide hydrolase activities determined in 27 subjects varied from 8.2 to 18.5 and from 3.5 to 17.0 pmol product formed per min per 10(7) cells, respectively. The mean coefficient of intraindividual variation, determined with three subjects measured four times each over the course of 18 days, was approximately 10% for both enzyme activities.

Benzo(a)pyrene metabolism by rat liver microsomes: effects of adding purified glutathione S-transferases A, B, and C.

We have examined the effects of adding glutathione and isolated cytosolic glutathione S-transferases A, B, and C to rat liver microsomes metabolizing benzo(a)pyrene. Addition of glutathione alone resulted in the conjugation of 15 to 20% of the total metabolites of benzo(a)pyrene, and this conjugation could be inhibited almost entirely by bromosulfophthalein (an inhibitor of glutathione S-transferases), indicating that it is catalyzed by the glutathione S-transferase present in microsomes. Addition of purified cytosolic glutathione S-transferases A, B, and C yielded about 30 to 40% conjugate formation. Analysis of metabolites by high-pressure liquid chromatography demonstrated that the formation of 4,5-diol of benzo(a)pyrene was decreased by at least 80% by conjugation and that the 7,8-diol was also decreased significantly (40 to 60%). In addition, it was found that glutathione S-transferase B is capable of conjugating benzo(a)pyrene 1,6- and 3,6-quinones.

Characterization of rat lung epoxide (styrene oxide) hydrase with a modified radioactive assay of improved sensitivity.

The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.

Characterization of the induction of drug-metabolizing enzymes by 2-acetylaminofluorene.

Changes in hepatic drug-metabolizing enzymes after intraperitoneal treatment of rats with 2-acetylaminofluorene have been investigated. This treatment was found to increase microsomal epoxide hydrolase to 762%, cytochrome P-450 to 143%, NADPH-cytochrome c reductase to 160%, cytochrome b5 to 171%, cytoplasmic DT-diaphorase to 229% and soluble glutathione S-transferase activities to 200-250% of control values. These increases were time- and dose-dependent, being maximal after injection of 50 mg 2-acetylaminofluorene/kg body wt. once daily for 5 days. Enzyme markers for the plasma membrane, mitochondria, lysosomes and the soluble cytoplasm were not affected by treatment with 2-acetylaminofluorene. The present study indicates that this induction is different from that obtained with phenobarbital and 3-methylcholanthrene and more closely resembles that seen with trans-stilbene oxide.

The proliferation of hepatocytes and the lipid composition of the endoplasmic reticulum after induction of drug-metabolizing enzymes with trans-stilbene oxide.

Three aspects of the induction of drug-metabolizing enzymes brought about by trans-stilbene oxide have been investigated. (1) The liver hypertrophy in rats treated with trans-stilbene oxide was found to result solely from an increase in the number of cells in this organ, without any increase in the size of each individual cell. (2) Administration of trans-stilbene oxide also produces a 27% increase in the phospholipid content of the hepatic endoplasmic reticulum, i.e., a limited proliferation of this organelle occurs. (3) Furthermore, induction causes changes in the lipid composition of the endoplasmic reticulum. The cholesterol content is decreased, the relative content of sphingo-myelin is also lowered, and a number of changes in the fatty-acid composition occur as well. All of these effects would tend to increase the fluidity of the phospholipid bilayer of the endoplasmic-reticulum membrane and may thus affect drug metabolism.

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver have been investigated. After perfusing the lung to remove contaminating blood, this organ was found to have an apparent concentration of glutathione (2mM) which is approx. 20% of that found in the liver. Both organs contain very low levels of glutathione disulfide. Neither phenobarbital nor methylcholanthrene had a significant effect on the levels of reduced glutathione in lung and liver. In addition, the activities of some glutathione-metabolizing enzymes--glutathione reductase and glutathione S-transferase activity assayed with four different substrates--were observed to be 5-to 60-fold lower in lung tissue than in the liver.

Effect of inducers of drug-metabolizing enzymes on glutathione reductase and glutathione peroxidase in rat liver.

Cytosolic glutathione reductase activity of rat liver was shown to increase to about 250% of control values after treatment of the animals by intraperitoneal injections of trans-stilbene oxide. The time course and dose-response relationship of the induction brought about by trans-stilbene oxide were determined. The increase of activity was accompanied by a similar increase of protein precipitable by antibodies to rat liver glutathione reductase. These results strongly indicate that increase of glutathione reductase activity in response to treatment with this inducer is the result of true induction. Phenobarbital and 3-methylcholanthrene increased the activities per mg protein in the cytosol fraction by 80 and 24%, respectively. Glutathione reductase was purified to homogeneity from rats treated with trans-stilbene oxide. The molecular and kinetic properties investigated were not significantly different from those of the enzyme from control animals. Selenium-dependent glutathione peroxidase was not induced in the hepatic cytosol of animals treated with trans-stilbene oxide.

Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate.

Intact microsomal vesicles from rat liver were subjected to combined treatment with trypsin and an unspecific protease and were also examined after reaction with the chemical probe p-diazobenzene sulfonate. In addition, the latency of various enzymes in intact microsomal vesicles has been investigated. All microsomal electron transport enzymes studied, i.e. NADH-ferricyanide and cytochrome c reductases, cytochrome b5, NADPH-cytochrome c reductase and cytochrome P-450, were either solubilized or inactivated by one or both treatments. The experimental data indicate that UDPglucuronyl-transferase is also localized at the outer surface of microsomes. In contrast, a number of hydrolytic enzymes are apparently located inside the permeability barrier of the membrane and presumably at the inner surface. Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats. This indicates that the enzyme molecules are not relocated after their insertion into the membrane.

Isolation of myelin bodies from the kidney cortex of gentamicin-treated rats.

Myelin bodies have been isolated from the kidney cortex of gentamicin-treated rats (100 mg gentamicin sulfate/kg body weight i.p. twice daily for 3 days) by a simple procedure involving differential centrifugation followed by equilibrium density centrifugation on a discontinuous sucrose gradient. Electron microscopy and assay of acid phosphatase suggest that the myelin bodies were obtained in virtually quantitative yield, essentially uncontaminated by other cellular structures and relatively intact. The method developed here may also prove applicable for the isolation of myelin bodies arising in connection with other drug treatments and may provide information on a number of toxic side-effects of clinical importance.

Energy levels in resting and mitogen-stimulated human lymphocytes during treatment with FK506 or cyclosporin A in vitro.

By employing microcalorimetry to assess overall metabolic activity in combination with other assays for specific metabolic events, we have investigated the influence of cyclosporin A and FK506 on the metabolic status of resting and mitogen-stimulated human peripheral lymphocytes. Both cyclosporin A and FK506 significantly reduced heat output from resting lymphocytes. This reduction could not be correlated with effects on DNA synthesis, lactate production, ATP levels or mitochondrial uptake of Rhodamine 123. These two drugs also potently reduced the increase in heat output seen during mitogen stimulation of lymphocytes. Both cyclosporin A and FK506 also prevented the increase in DNA synthesis, lactate production and ATP levels seen in response to mitogen stimulation. The increase in mitochondrial uptake of Rhodamine 123 during blastoid transformation was significantly reduced only by cyclosporin A. We ascribe the major part of the effects of these compounds to inhibition of the glycolytic pathway in both resting and mitogen-stimulated lymphocytes. These results indicate that the immunosuppressants cyclosporin A and FK506 exert other effects on lymphocytes than their well-established inhibitory action on calcineurin.

Purification and initial characterization of microsomal epoxide hydrolase from the human adrenal gland.

Microsomal epoxide hydrolase from the human adrenal gland was purified to a high degree of homogeneity in 10% overall yield using sequential chromatography on DE-52, FPLC Mono Q and FPLC Superose columns. The fact that the overall purification was only 7.3-fold indicates that approx. 14% of the total microsomal protein consisted of this enzyme, a uniquely high value. The human adrenal enzyme was found to resemble rat liver microsomal epoxide hydrolase closely in a number of respects, including molecular weight, N-terminal amino-acid sequence and response to low-molecular weight ligands. However, rabbit antibodies directed against human adrenal microsomal epoxide hydrolase crossreacted only weakly with the corresponding rat liver protein. The relatively high levels of microsomal epoxide hydrolase in the human adrenal gland suggest that this enzyme may be of particular importance in this tissue. However, very little cytochrome P-450-catalyzed metabolism of xenobiotics has been demonstrated in the human adrenal and our present results speak against the involvement of microsomal epoxide hydrolase in the steroid metabolism of this gland. Thus, the function of this enzyme in the human adrenal is enigmatic.

Synergistic induction of acyl-CoA oxidase activity, an indicator of peroxisome proliferation, by arachidonic acid and retinoic acid in Morris hepatoma 7800C1 cells.

Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 microM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxy-arachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.1-0.8 microM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 microM all-trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all-trans-retinoic acid. When all-trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all-trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR alpha activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR alpha heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic acid as is the same process caused by arachidonic acid.

Purification and characterization of the major microsomal cytochrome P-450 form induced by trans-stilbene oxide in rat liver.

The major form of microsomal cytochrome P-450 induced by trans-stilbene oxide in the liver of male Sprague-Dawley rats was purified and characterized, and compared with the isolated cytochrome P-450 B2 forms from phenobarbital- and 3-methylcholanthrene-pretreated animals. The apparent subunit molecular weight of the trans-stilbene oxide-induced cytochrome was found to be 53 000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the absorbance maximum of the carbon monoxide complex of the ferrous cytochrome was 450 nm. Reconstitution of the N-demethylase activity towards three different substrates showed high and similar activities with the cytochrome P-450 B2 forms from trans-stilbene oxide or phenobarbital-treated rats, with one exception. Amino-acid analysis also showed a very high degree of similarity between these two forms. Upon proteinase treatment with three different proteinases the trans-stilbene oxide-induced cytochrome demonstrated in each case a peptide pattern identical to that obtained with the phenobarbital-induced B2 form. Furthermore, both forms are completely immunologically cross-reactive. We therefore conclude from these experiments that the liver microsomal P-450 B2 from trans-stilbene oxide and phenobarbital-treated rats are very closely related, if not identical.

Induction of glutathione S-transferases A, B and C in rat liver cytosol by trans-stilbene oxide.

The cytoplasmic glutathione S-transferase activity of rat liver has been shown to increase to 300--400% of control values after treatment of the animals with trans-stilbene oxide and this phenomenon has been further characterized in the present study. Quantitative immunological determinations showed that the content of glutathione S-transferases A, B and C together constituted 4.5% of the soluble proteins in the hepatic cytoplasm of untreated rats. The content rose to 12.9 and 17.4% after treatment with trans-stilbene oxide or a combination of trans-stilbene oxide, 3-methylcholanthrene and phenobarbital, respectively. It was demonstrated that the cytosolic fraction from induced liver contains 4.2 times as much antigen which can be precipitated with antiglutathione S-transferase B antiserum as does control cytosol. Antiglutathione S-transferase C, which intereacts with transferases A and C, precipitates 3.3 times as much protein from the induced cytosol compared with control. Crossed immunoelectrophoresis and purification demonstrated that both A and C are increased in amount after treatment with trans-stilbene oxide. Thus, cytosolic glutathione S-transferases A, B, and C in liver are all induced by treatment of rats with trans-stilbene oxide. Immunological crossreaction, similar behavior during chromatography on CM-cellulose and hydroxyapatite and similar specific activities suggest that the control and induced enzymes are essentially identical, trans-Stilbene oxide was found to serve as a relatively poor second substrate for glutathione S-transferases A, B and C and can thus be said to cause substrate induction of these enzymes.

Induction of drug-metabolizing systems and related enzymes with metabolites and structural analogues of stilbene.

trans-Stilbene oxide has been found to be a new type of inducer of drug-metabolizing systems. In order to identify the true inducer and to determine the structural requirements for induction, rats were treated with metabolites and structural analogues of stilbene. Subsequently, hepatic levels of cytochrome P-450, microsomal epoxide hydrolase, and cytoplasmic glutathione S-transferase were assayed. All three enzymes were induced by cis- and trans-stilbene and cis- and trans-stilbene oxide. In addition, epoxide hydrolase and glutathione S-transferase activities were induced by benzoin and benzil. In contrast, the diols and benzoic acid had little, if any, effect. The main conclusions drawn from these findings are that: (1) trans-stilbene oxide itself seems to be the inducer of drug-metabolizing enzymes; and (2) benzil is more selective as an inducer of epoxide hydrolase than is trans-stilbene oxide. Attempts to induce epoxide hydrolase with other structural analogues of stilbene led to the following conclusions: (1) two phenyl rings are required for induction; (2) the induction is not as great if the rings are substituted or one of the ring carbon atoms is replaced by a nitrogen; (3) a carbon bridge between the phenyl groups generally results in a greater induction, especially if the bridge contains an epoxy group or one or two keto groups.