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.

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat.

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.

Preparation and characterization of subcellular fractions from the head kidney of the Northern pike (Esox lucius), with particular emphasis on xenobiotic-metabolizing enzymes.

The present study was designed to prepare and characterize subcellular fractions from the head kidney of the Northern pike (Esox lucius), with special emphasis on the preparation of a microsomal fraction suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation as well as the recovery of different cell components was determined using both enzyme markers and morphological criteria. Finally, the subcellular distributions of several drug-metabolizing enzymes (NADPH-cytochrome c reductase, NADH-ferricyanide reductase, glutathione transferase, epoxide hydrolase) were determined. With the exception of NADPH-cytochrome c reductase, the subcellular distributions obtained here for drug-metabolizing and marker enzymes closely resembled those reported for rat liver. NADPH-cytochrome c reductase was apparently partially solubilized here from microsomal vesicles by an endogenous protease, which reduced its usefulness as a marker enzyme and raises questions concerning the measurement of activities catalyzed by the cytochrome P-450 system in these subfractions. In other respects the microsomal fraction prepared here from the pike head kidney seems well-suited for studies of drug metabolism.

Characterization of soluble glutathione transferase activity in resting mononuclear leukocytes from human blood.

Glutathione transferase activity in the soluble fraction of resting human mononuclear leukocytes was measured and characterized using [3H] trans-stilbene oxide as a substrate. Because of the low activity of this enzyme in these cells, a published assay procedure developed for rodent liver was slightly modified to improve its sensitivity: the substrate was highly radiolabeled (2 Ci/mmole) and carefully purified, and the incubation time was extended to 30-60 min. The activity measured was linear with cell density up to at least 6 million cells. Soluble glutathione transferase activity measured in this manner has a pH optimum around 7.4 and an optimal temperature of 40 degrees. This activity could be measured in lymphocytes, monocytes, granulocytes, erythrocytes and platelets, but not in plasma. From these measurements it could be calculated that lymphocytes account for somewhat more than half of the total activity in the mononuclear leukocyte fraction and that monocytes account for the rest. The intraindividual variation in soluble glutathione transferase activity towards trans-stilbene oxide in the mononuclear leukocyte fraction from different subjects was only about 10%, whereas the interindividual variation in this same activity was 15-fold. An explanation for this relatively large interindividual variation is now being sought.

Effects of tobacco and tobacco smoke constituents on cell multiplication in vitro.

Ascites sarcoma BP8 cells, cultured in suspension in vitro were used as a general toxicity test system for tobacco and tobacco smoke constituents. Some 250 compounds, representative of these materials, were examined by exposing cells to different concentrations of these constituents and measuring the inhibition of culture growth, which was related to corresponding effects encountered for positive standards. When employing the present cell toxicity test system possible effects of factors such as penetration, distribution and microsomal metabolism of the compounds studied, are not taken into account. The most active constituents were found to be unsaturated aldehydes and ketones, phenols and indoles. The good correlation obsered between functional groups and toxicity permits, within the range of functionalities studied, prediction of the toxicity for a compound of known structure.

Long-term fate of [14C]nicotine in the mouse: retention in the bronchi, melanin-containing tissues and urinary bladder wall.

N-methyl-14C and 2'-14C-labelled nicotine were used for whole-body autoradiographic distribution studies on C57BL- and NMRI-mice. Radioactivity was retained in the melanin-containing tissues, in the bronchial walls, and in the urinary bladder wall, up to 1 month after administration. The activity levels in the bronchi decreased faster if [2'(14)C] nicotine was used. Quantitative measurements of the retention of the 2 14C-labelled nicotine preparations confirmed the autoradiographic findings. It is proposed that nicotine is N-demthylated in the bronchial mucosa, the off-coming methyl group being incorporated into the cell constituents of the mucosa. Thin-layer chromatographic studies showed that no nicotine was present in the lungs after 24 h. In melanin, however, only unmetabolized nicotine was found from 4 h on. Some reactive nicotine metabolites may be responsible for the retention in the urinary bladder wall. Also in the full-term fetuses radioactivity accumulated in the pigmented eyes and in the respiratory tract. The accumulation and long-term retention of nicotine in the melanin-containing structures might accelerate the development of drug-induced or senile changes in these tissues. The retention in the urinary bladder wall persisted even after rinsing. This may indicate an accumulatory mechanism worth considering in the pathogenesis of urinary bladder cancer.

Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid.

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.

[Occlusions and perforations caused by carcinoma of the colon and rectum]. / Occlusioni e perforazioni da carcinoma del colon e del retto

A group of 137 patients observed at the Emergency Surgery and First Aid Division of the Fatebenefratelli Hospital of Milan between 1971 and 1974 is examined. The patients all presented a clinical picture of occlusion and/or perforation of the colon and rectum due to carcinoma, requiring emergency surgery. The clinical material examined is analysed and the surgical treatment adopted and the results obtained reported. Post-operative mortality was 16.8% in cases of occlusion, 21.6% in cases of perforation, and 36.4% in mixed cases. 63 patients, i.e. 46%, were subjected to more or less extensive resection and the mortality in this group was 11.2%; in the remaining 74 patients, ax only palliative measures were possible and mortality was 28.3%. It is observed, finally, that among emergency colon operations, right colectomy carried out immediately offers encouraging results, while resection of the left colon, apart from certain special cases, should be preceded by decompressive colostomy.

[Our experience with emergency surgical treatment of acute cholecystitis]. / La nostra esperienza nel trattamento chirurgico d'ungenza delle colecistiti acute.

A clinical series of 580 patents (318, F, 199 M) personally observed at the Emergency Surgery and First Aid Division of the Fatebenefratelli and Ophthalmic Hospital Board of Milan between 1975 and 1980, and suffering from acute inflammation of the bile ways (gall bladder empyemas, acute cholecystitis, gangrene of the gall bladder, haemobilia due to gall bladder puncture), has been examined. Of these patients, 558 were subjected to surgery between 12 hours and 6 days after admittance. Operated patients are subdivided into 4 groups on the basis of their anatomo-pathological form and the average time interval between admittance and intervention. Critical examination shows that their behaviour with respect to acute inflammatory forms of the gall bladder can be split up as follows: 1) immediate surgery (within 12 hours) for empyematous and/or punctured forms; 2) emergency surgery (within 2 days of admittance) for cases with certain diagnosis backed up by historical and X-ray data pointing to calculosis of the gall bladder; 3) early surgery (within 3 days) for cases with certain diagnosis but without prior X-ray documentation; 4) deferred surgical intervention (within 6 days) for patients without X-ray documentation and in whom immediate medical treatment leads to a rapid improvement in the clinical picture. The very good clinical results obtained and the observation of a low mortality and morbility index (comparable to those of surgery of choice) suggest that early surgery is certainly the therapy of choice when dealing with acute cholecystitis.

The effect of tobacco smoke condensate on the growth and longevity of human diploid fibroblasts.

Human embryonic diploid lung fibroblasts were exposed to various fractions of cigarette smoke condensate over their in vitro life-time. Most fractions were toxic at a concentration of 50 microgram/ml, with the exception of the strong acid and one water soluble basic fraction, which stimulated growth and increased longevity significantly at this concentration. Most fractions produced no effect on cell growth at 10 microgram/ml, with the exception of another basic fraction which inhibited growth at 1 microgram/ml. Nicotine had no apparent effect on growth and longevity at 50 microgram/ml. The neutral fraction containing the polynuclear hydrocarbon carcinogens produced normal growth and longevity at 10 microgram/ml. No cell transformations were observed.

Induction of different isozymes of cytochrome P-450 and of microsomal epoxide hydrolase in rat liver by 2-acetylaminofluorene and structurally related compounds.

The amounts of five different forms of cytochrome P-450 and of microsomal epoxide hydrolase were determined immunochemically in rat liver microsomes before and after treatment of the animals with 2-acetylaminofluorene and 15 structurally related compounds. The amount of cytochrome P-450c was found to be increased about 60-fold after treatment with 2-aminofluorene and 3-aminofluorene. Administration of 1-aminofluorene, 4-aminofluorene, 2-acetylaminofluorene and nitrofluorene increased this isozyme about 15-19 times. 2-Aminofluorene was found to inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a cytoplasmic receptor 50% at a concentration of 3.12 microM, while no such inhibition could be detected with 2-acetylaminofluorene. Induction of ethoxyresorufin O-deethylase activity was found to be highly correlated (+0.95) with the induction of cytochrome P-450c. Also correlated with the induction of this form was the amount of cytochrome P-450d (+0.84), which could be maximally increased about fourfold. Cytochromes P-450b + e were induced by 2-acetylaminofluorene, 4-acetylaminofluorene and fluorene (about tenfold), while 4-aminofluorene and 4-acetylaminofluorene were found to elevate cytochrome P-450PB/PCN-E about threefold. Microsomal epoxide hydrolase was induced by many of the compounds tested, with 2,7-diaminofluorene, 2,7-diacetylaminofluorene, 2-acetylaminofluorene and 2-(N-hydroxy)acetylaminofluorene being the most potent. No correlation of the induction of this enzyme with the induction of any isozyme of cytochrome P-450 was observed.

Synthesis of a glucoheptaose and a glucooctaose that elicit phytoalexin accumulation in soybean.

The glucoheptaose 1 and the glucooctaose 2 have been synthesized using unambiguous methods. The former is identical with an elicitor-active heptasaccharide obtained from partially hydrolyzed mycelium of Phytophthora megasperma f. sp. glycinea. The octasaccharide is also elicitor active, although to a lesser extent than the heptasaccharide. 1:R = H; 2:R = beta-D-Glcp. (Formula: see text)

Comparison of the structures and elicitor activities of a synthetic and a mycelial-wall-derived hexa(beta-D-glucopyranosyl)-D-glucitol.

The elicitor activity and structural characteristics of chemically synthesized hepta- and octa-beta-D-glucopyranosides were compared with the same properties of an elicitor-active mycelial-wall-derived hexa(beta-D-glucopyranosyl)-D-glucitol. The specific elicitor activities, retention times on reversed-phase liquid chromatography columns, glycosyl-linkage compositions, 1H NMR analyses, and glycosyl-sequence analyses of the synthetic and mycelial-wall-derived hexa(beta-D-glucopyranosyl)-D-glucitols were indistinguishable. This work provided conclusive proof that elicitor activity was associated with the structure proposed (Sharp, J. K., McNeil, M., and Albersheim, P. (1984) J. Biol. Chem. 259, 11321-11336) for the elicitor-active mycelial-wall-derived hexa(beta-D-glucopyranosyl)-D-glucitol.

Structure-activity relationships of oligo-beta-glucoside elicitors of phytoalexin accumulation in soybean.

The abilities of a family of chemically synthesized oligo-beta-glucosides, ranging in size from hexamer to decamer, to induce phytoalexin accumulation in soybean cotyledons were investigated to determine which structural elements of the oligoglucosides are important for their biological activity. The results of the biological assays established that the following structural motif is necessary for the oligo-beta-glucosides to have high elicitor activity: [formula; see text] The branched trisaccharide at the nonreducing end of the oligoglucosides was found to be essential for maximum elicitor activity. Substitution of either the nonreducing terminal backbone glucosyl residue or the side-chain glucosyl residue closest to the nonreducing end with glucosaminyl or N-acetylglucosaminyl residues reduced the elicitor activity of the oligoglucosides between 10-fold and 10,000-fold. Elicitor activity was also reduced 1000-fold if the two side-chain glucosyl residues were attached to adjacent backbone glucosyl residues rather than to glucosyl residues separated by an unbranched residue. In contrast, modifications of the reducing terminal glucosyl residue of an elicitor-active hepta-beta-glucoside by conjugation with tyramine and subsequent iodination had no significant effect on the elicitor activity of the hepta-beta-glucoside. These results demonstrate that oligo-beta-glucosides must have a specific structure to trigger the signal transduction pathway, which ultimately leads to the de novo synthesis of phytoalexins in soybean.