Biochemical effects of dietary intake of different broccoli samples. II. Multivariate analysis of contributions of specific glucosinolates in modulating cytochrome P-450 and antioxidant defense enzyme activities

Dietary broccoli exposure modulates various cytochrome P-450 (CYP)-associated activities and antioxidant defense enzyme activities in liver, colon, and kidney of rats. We present an analysis by the partial least-square method (PLS) of the contribution of single glucosinolates in modulating xenobiotic metabolizing and antioxidant defense enzyme activities. Generally, modulation of colonic enzyme activities was well described (58% to 75%) by models consisting of 3 principal components (PCs). The indolyl glucosinolates were not the only major contributors to the regulation of colonic 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD) activities, as would be expected from results of previous experiments testing the pure compounds, glucobrassicin (GB), neoglucobrassicin (NeoGB), and 4-methoxyglucobrassicin (4-MeOGB). In hepatic and renal microsomes, the modulation of enzyme activities could be partly described for hepatic and renal 7-pentoxyresorufin O-deethylase (PROD) activities (42% to 44%, 3 to 4 PCs), hepatic superoxide dismutase activity (45%, 2 PCs), and renal glutathione peroxidase (GSH Px) and glutathione reductase (GSSG Red) activities (43%, 3 PCs). These results indicate that substances other than glucosinolates in the complex mixtures modulate hepatic EROD, MROD, GSH Px, and GSSG Red activities or that the active glucosinolate metabolites vary in their systemic disposition.     

Effects of aging and long-term caloric restriction on hepatic microsomal monooxygenases in female fischer 344 rats: Alterations in basal cytochrome P-450 catalytic activities

The effects of aging and caloric restriction on uninduced levels of hepatic microsomal cytochrome P-450s and specific P-450-related catalytic activities were evaluated in female Fischer 344 rats. Microsomes were isolated from livers of ad libitum (AL) fed rats 1, 3, 6, 16 and 26 months of age, and from calorie-restricted (CR) rats 6, 16 and 26 months of age. The recovery of microsomal protein was higher in CR than AL rats at 6, 16 and 26 months of age. Between ages 3 and 26 months there was a gradual decline in recovered microsomal P-450, but the differences between successive age categories were not significant. A different trend was observed for total cytochrome P-450 per liver. AL rats showed a 2-fold decline in P-450/liver between 6 and 26 months of age, while P-450/liver of corresponding CR rats increased 1.7-fold. Neither cytochrome b₅ nor NADPH cytochrome C reductase varied consistently as a function of increased age, with all age groups showing greater reductase activity for CR rats than for AL rats. Phenobarbital (PB) and methylcholenthrene (MC) inducible cytochrome P-450 catalytic activities (pentoxy [PROD] and ethoxy [EROD] resorufin O-deethylases) exhibited similar age-related alterations that were distinctly different from the two other enzymes examined. Both PROD and EROD showed dramatic declines in activity between 1 and 3 months of age, with a slight increase in activity at 6 months and maintenance of the activity levels through 26 months. Benzphetamine N-demethylase (BND) and aniline p-hydroxylase (APH) activities were also altered as a function of increased age, with CR rats exhibiting increased isozyme activity above that seen for aging AL rats. Calorie restriction appears to have a modulating effect, compensating for an age-related decline of hepatic microsomal monooxygenase activity in female Fischer 344 rats.     

Effects of frying oil and Houttuynia cordata thunb on xenobiotic-metabolizing enzyme system of rodents

AIM: To evaluate the effects of frying oil and Houttuynia cordata Thunb (H. cordata), a vegetable traditionally consumed in Taiwan, on the xenobiotic-metabolizing enzyme system of rodents. METHODS: Forty-eight Sprague-Dawley rats were fed with a diet containing 0%, 2% or 5% H. cordata powder and 15% fresh soybean oil or 24-h oxidized frying oil (OFO) for 28 d respectively. The level of microsomal protein, total cytochrome 450 content (CYP450) and enzyme activities including NADPH reductase, ethoxyresorufin 0-deethylase (EROD), pentoxyresorufin 0-dealkylase (PROD), aniline hydroxylase (ANH), aminopyrine demethylase (AMD), and quinone reductase (QR) were determined. QR represented phase Ⅱ enzymes, the rest of the enzymes tested represented phase Ⅰ enzymes. RESULTS: The oxidized frying oil feeding produced a significant increase in phase Ⅰ and Ⅱ enzyme systems, including the content of CYP450 and microsomal protein, and the activities of NADPH reductase, EROD, PROD, ANH, AMD and QR in rats (P<0.05). In addition, the activities of EROD, ANH and AMD decreased and QR increased after feeding with H. cordata in OFO-fed group (P<0.05). The feeding with 2% H. cordata diet showed the most significant effect. CONCLUSION: The OFO diet induces phases I and II enzyme activity, and the 2% H. cordata diet resulted in a better regulation of the xenobiotic-metabolizing enzyme system.     

Pathogenic aspects of pyogenic liver abscess associated with experimental schistosomiasis.

Schistosomiasis mansoni infection that occurs concurrently with Staphylococcus aureus bacteremia favors the formation of pyogenic liver abscess. The present experimental study in mice evaluated the following aspects of the relationship between infection with Schistosoma mansoni and liver abscess caused by S. aureus: a) the role of the eggs of S. mansoni in the genesis of the abscesses; b) the influence of different phases of schistosomiasis in the development of liver abscesses; and c) the effect of the treatment of schistosomiasis on the development of the abscesses. Macroscopic and histopathological study showed multiple liver abscesses around granulomas of S. mansoni in the acute and chronic phases of schistosomiasis. Treatment of acute schistosomiasis before experimentally-induced bacteremia did not prevent the formation of liver abscess. The study findings indicate that granulomas around S. mansoni eggs and worms lodged in the liver provide a focus and substrate for pyogenic abscesses caused by S. aureus.     

Effect of murine schistosomiasis on hepatic cytochrome P-450 and microsomal protein

Experimental hepatic schistosomiasis was produced in CBA mice using a local strain of S. mansoni. A comparative study of the hepatic concentrations of cytochrome P-450 and microsomal protein in the control and infected animals was carried out. S. mansoni infection significantly (P less than 0.05) reduced the liver cytochrome P-450 and microsomal protein. This suggests impairment of drug metabolism in the liver of infected animals. The study calls attention to the possible clinical and pharmacokinetic implications of the late severe S. mansoni infection of the liver in man.     

Comparison of immune responses of Schistosoma mansoni-infected mice with distinct chronic forms of the disease

BACKGROUND: Schistosoma mansoni-infected mice tend to present with either one of two different hepatic pathological patterns during chronic infection: periportal fibrosis (PF) with portal concentration of periovular granulomas and fibrosis or isolated granulomas (IG), with scattered periovular granulomas within the liver. These are models for the two clinical presentations of schistosomiasis, the severe hepatosplenic and the mild intestinal forms. In the present work, we examined the relationship between the development of these histopathological aspects and immunological markers in S. mansoni-infected mice. Although BALB/c mice with PF and IG had similar egg numbers in the liver, PF mice had higher liver collagen contents than mice with IG. Cultured spleen cells from mice with PF and IG had similar proliferation 20 and 40 weeks after S. mansoni infection upon stimulation with parasite egg antigen (SEA) or mitogen (Con A). Production of IL-4 upon SEA stimulation was higher in cell cultures from mice with PF, whereas IL-5 and IFN-gamma levels were not statistically different between PF and IG groups. Mice with IG had similar serum concentrations of total IgE and anti-SEA IgG1, IgG2a, IgG2b and IgG3 compared to sera from PF mice. Levels of IgG1 and IgG2a antibodies were the highest and the lowest detected, respectively. In conclusion, isogenic BALB/c mice infected with S. mansoni that develop periportal fibrosis or isolated granulomas have similar immunological patterns despite the two pathologic forms of schistosomal liver fibrosis.     

Inducibility of the hepatic drug-metabolizing capacity of mice infected with Schistosoma mansoni.

The activities of some hepatic microsomal drug metabolizing enzymes, which are markedly depressed in mice infected with Schistosoma mansoni, can be increased by treatment with phenobarbital or 3-methylcholanthrene. Administration of these compounds to infected mice increased the capacity of the liver to metabolize drugs up to the maximum level inducible in non-infected animals. However, the increased hepatic microsomal mass, reflected in glucose 6-phosphatase activities and cytochrome b5 levels, observed in schistosome-infected mice, was not increased further by the same treatment. The changes in the activities of several drug metabolizing enzymes in vitro were confirmed in vivo by determination of hexobarbital-induced sleeping time and zoxazolamine-induced paralysis duration.     

Differences in Hepatic Microsomal Cytochrome P-450 Isoenzyme Induction by Pyrazole, Chronic Ethanol, 3-Methylcholanthrene, and Phenobarbital in High Alcohol Sensitivity (HAS) and Low Alcohol Sensitivity (LAS) Rats

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical effects of dietary intakes of different broccoli samples. I. Differential modulation of cytochrome P-450 activities in rat liver, kidney, and colon

Modulation of xenobiotic metabolism, including cytochrome P-450 (CYP) enzyme activities, due to dietary intakes of cruciferous vegetables, has been described in animals and humans, and the induction of CYP1A enzymes is suggested mainly to be related to the content of indolyl glucosinolates in these vegetables. The aim of the present study was to evaluate the effects on specific CYP activities of various broccoli samples containing different levels of glucosinolates. Groups of rats were fed 1 of 8 broccoli samples from 2 cultivars grown at different conditions. Thirteen different glucosinolates were quantified. The content of the 4 major glucosinolates, glucoraphanin (GRAP), glucoiberin, glucobrassicin (GB), and neoglucobrassicin (NeoGB) varied 5.6-, 2.7-, 3.2-, and 6.6-fold, respectively, among the broccoli samples. Dietary broccoli induced the CYP1A enzyme activities, 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD), in rat liver, weakly in colon, but not in kidney. In concordance, the hepatic metabolism of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) to the proximate carcinogen N-OH-PhIP, a CYP1A-related activity, was enhanced by broccoli. The 7-pentoxyresorufin-O-depenthylase (PROD) activity, an assay for CYP2B1/2, was weakly induced in colon and kidney but not in liver by broccoli. The 2 beta-OH- and 6 beta-OH-testosterone hydroxylase activities were induced in liver microsomes, showing that broccoli increased CYP3A activity. The observed modulations of CYP activities depended clearly on the broccoli sample used, and significantly different responses were observed for different cultivars and growth conditions. These results indicate that modulation of CYP metabolism by broccoli may vary significantly in humans as well, as the content of glucosinolates and other active substances also varies between commercially available broccoli samples. The different effects depending on the vegetable sample eaten have to be considered in future experiments and dietary recommendations.     

Immunopathology of Schistosoma japonicum infection in athymic mice

Athymic (nu/nu) mice and heterozygous littermate controls (nu/+) were examined 7 and 10 weeks after infection with 10 cercariae of Schistosoma japonicum. Schistosome infection developed normally in both groups of mice and eggs were produced in normal numbers. Nu/nu mice developed small circumoval granulomas with minimal fibrosis while nu/+ mice developed large fibrotic granulomas. Unlike the mononuclear responses to S. mansoni eggs at 7 weeks, those to S. japonicum often were abscess like with narrow rims of liver cell necrosis or microvesicular fatty change. However, evolving granulomas in nu/+ mice were enriched with eosinophils, epithelioid macrophages, immature granulocytes and plasma cells, all scarce in the corresponding nu/nu lesions as were fibroblasts and collagen fibres, thus accounting for their smaller mean size and better healing. Our aggregate evidence shows that normal granuloma formation and cellularity in S. japonicum infection is controlled by T-cells as is the case for S. mansoni, and not by antibodies or immune complexes.     

青蒿琥酯对小鼠曼氏血吸虫虫卵肉芽肿形成的影响

目的通过口服青蒿琥酯,观察其对曼氏血吸虫感染小鼠肝脏肉芽肿形成的影响。方法小鼠经尾部接触感染曼氏血吸虫尾蚴后,经不同时间、不同剂量喂服青蒿琥酯,治疗后2～6周解剖,取肝脏作石蜡切片,进行H.E.染色,显微镜下作病理学观察,并测定虫卵肉芽肿的面积。结果未给药对照组虫卵肉芽肿由大量炎性浸润细胞及间质成纤维细胞组成,服药组肝切片未见虫卵肉芽肿或虫卵肉芽肿面积明显小于对照组,肉芽肿周围少量炎性细胞浸润。100mg/kg组肉芽肿面积与对照比较无显著性差异(P>0.05)。但肉芽肿面积比对照减小36.9%。300、500mg/kg组病理切片未查到虫卵和肉芽肿。不同虫龄服药组肉芽肿面积与对照比较均有显著性差异(P<0.05～P<0.01)。其中21、22d服药组肝切片未查到虫卵和肉芽肿。结论青蒿琥酯能抑制虫卵肉芽肿的形成,降低虫体产卵率并减少形成的肉芽肿对肝脏的损害。     

Alterations in influence of granuloma-derived cytokines on fibrogenesis in the course of murine Schistosoma mansoni infection

Schistosomiasis is the main cause of hepatic fibrosis worldwide, yet its pathogenesis remains unknown. We previously reported that conditioned medium from cultures of hepatic egg granulomas (isolated from mice acutely infected with Schistosoma mansoni) can stimulate fibroblast proliferation and matrix production in vitro. We have proposed that initiation of hepatic fibrosis in this infection might be under the control of granuloma-derived cytokines. We now report that conditioned medium from cultures of schistosomal egg granulomas isolated from liver of chronically infected mice has reduced fibrogenic activity compared with medium from cultures of granulomas obtained from more acutely infected mice. In related studies, we adoptively transferred splenocytes from infected mice and examined the fibrogenic activity in culture supernatants of hepatic egg granulomas isolated from the recipients. Those prepared from recipients of splenocytes from chronically infected mice contained substantially less fibrogenic activity than did those from recipients of splenocytes of acutely infected mice. These findings suggest that the fibrogenic influence of schistosomal egg granuloma products decreases during the course of chronic murine S. mansoni infection. Furthermore, our preliminary findings suggest that immunoregulatory cells may be responsible for the down-regulation of this influence. Previous observations indicating that in murine schistosomiasis hepatic collagen and hyaluronate synthesis and deposition are elevated in acute but not chronic infection might be explained on the basis of our observations.     

Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report.

beta-Aminopropionitrile (BAPN) is an inhibitor of the lysyl oxidase required for cross-link formation in collagen maturation. The efficacy of BAPN, alone or in association with the anti-schistosomal drug, praziquantel (PZQ), was primarily assessed by measuring the reduction in liver and intestinal egg loads in murine schistosomiasis mansoni. Depending on the treatment group (PZQ, BAPN, BAPN + PZQ), organ-specific effects were observed using microscope image analysis. Most notable was the relatively small size of granulomas in the livers of BAPN-treated mice, which contrasted with the relatively large size and irregular shape of the granulomas in the intestinal tissues of these mice. Mice treated with the combination of BAPN and PZQ had decreased liver and spleen weights, and a significant reduction in the number of eggs trapped in both the liver (86%) and the intestine (99.1%), compared with untreated mice and those given PZQ alone. The lowest number of living eggs/g of tissue in both the liver and intestine was recorded in the combined BAPN + PZQ-treated group. These results suggest that the concurren treatment of infected mice with PZQ and BAPN enhances the release of eggs trapped in the intestine and also results in a significant reduction of liver egg load. The mechanism by which BAPN reduces the number of liver granulomas in PZQ-treated mice is currently being investigated.     

GOLD KINETICS UNDER LONG-TERM TREATMENT WITH GOLD(I) DISODIUM THIOMALATE: A COMPARISON IN THREE DIFFERENT MOUSE STRAINS

Following weekly i.m. injections of gold(I) disodium thiomalate(GST), mice of strains A.SW and C57BL/6 develop adverse immunereactions, whereas DBA/2 mice do not. We have studied the pharmaco-toxicokineticsof gold in these strains under chronic GST treatment. Our resultsindicate that the susceptible strains A.SW and C57BL/6 accumulatesignificantly higher gold concentrations in the liver and spleencompared to the resistant strain DBA/2. In the kidney of DBA/2mice, gold concentrations persisted at a plateau level, whereasin A.SW and, particularly, C57BL/6 mice early peaks of goldconcentrations were followed by a transient decrease, suggestiveof tubular toxicity. Whereas splenic T and B cells failed tocontain measurable gold concentrations in all three strains,splenic and peritoneal macrophages contained relatively highlevels, more so in the susceptible strain C57BL/6 than in theresistant DBA/2 strain. This finding is consistent with theconcept that macrophages play an important role in both theadverse and the beneficial effects of gold drugs. KEY WORDS: Anti-rheumatic drugs, Gold sodium thiomalate, Genetics, Inbred mouse strains, Macrophages, Pharmacokinetics, Toxicokinetics.     

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 microCi of [(14)C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 micromol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min.mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K(m) of 38+/-25 and 45+/-15 micromol/L, whereas the K(m) for retigabine N-glucuronidation by human liver microsomal fractions was 145+/-39 micromol/L. Furthermore, a V(max) of 1.2+/-0.3 (nmol/[min.mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine-N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 micromol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of (14)C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.     

Mesenchymal target cell specificity of egg granuloma-derived fibroblast growth factor in schistosomiasis

We previously demonstrated that egg granulomas isolated from the liver of mice infected with Schistosoma mansoni secrete factors that stimulate fibroblast proliferation in vitro. Because this growth factor also stimulated thymocyte proliferation, we further investigated the mesenchymal target cell specificity of this factor. We now report that granulomas also secrete material that promotes growth of aortic smooth muscle and endothelial cells. We found that the activities that stimulate fibroblast and smooth muscle cell growth share similar physicochemical properties (Mr, 30-40 kilodaltons [kDa]; pI, approximately 6.5), a result suggesting that they are the same or related molecules. In contrast, fractions (Mr, less than 10 kDa; pI, 6.7-7.5) that maximally stimulated endothelial cells had minimal or no effects on the other two cell types. We postulate that soluble granuloma products might also stimulate in vivo proliferation of vascular smooth muscle and endothelial cells and contribute to the pathology of the intrahepatic vessels in schistosomiasis.     

Comparison of the hepatoprotective effects of immune cells and serum in Schistosoma mansoni-infected immunosuppressed mice

Immunosuppressed mice with heavy Schistosoma mansoni infections suffer from a severe hepatotoxicity reaction soon after the onset of infection patency, and this may be directly consequent upon the failure of the hosts to mount adequate granulomatous responses to embolized parasite eggs. Immune serum or immune peripheral lymphocytes from normal S. mansoni-infected immunologically intact donor mice were transferred to homologously-infected syngeneic T-cell deprived recipients to test the respective capacities of the transferred humoral or cellular immune effector elements to prevent hepatocellular damage and to reconstitute granuloma formation. Transferred immune serum was very effective in preventing liver cell damage, but did not significantly reconstitute the capacity to form granulomas in the recipients. In contrast, mice receiving immune spleen and mesenteric lymph node cells had their capacity to form granulomas around liver-bound eggs reconstituted, but lymphoid cell transfer was less effective in protecting against hepatocyte damage than serum transfer. Protection of host tissues may therefore not be the main role of the T-cell mediated S. mansoni egg granuloma.     

Light and electron microscopic studies on Schistosoma mansoni Granulomas of mouse livers following treatment with praziquantel

The disintegration of Schistosoma mansoni and their eggs was studied in the liver of mice after termination of the infection by praziquantel. Egg granulomas, which were already present at the time of treatment, attained their maximal diameter of 380 microns after 2-3 weeks. Within the following 5 weeks, granulomas very rapidly regressed in size to only 165 microns. Directly after treatment, worms were trapped in the liver where they were quickly invaded by granulocytes and subsequently phagocytosed within 3 weeks. Worm granulomas measured 700 and 900 microns after 3 and 8 weeks, respectively, but then regressed rapidly to only 550 microns after 12 weeks. Liver lesions appeared to regress more rapidly after praziquantel than after treatment with other schistosomicidal drugs.     

Effects of light and dark beer on hepatic cytochrome P-450 expression in male rats receiving alcoholic beverages as part of total enteral nutrition

BACKGROUND: Alcoholic beverages contain many congeners in addition to ethanol. Therefore, consumption of alcoholic beverages may have considerably different effects on expression of hepatic microsomal monooxygenases than the relatively selective induction of cytochrome P-450 (CYP) 2E1 observed after consumption of pure ethanol. METHODS:: In the current study, we compared the effects of two beers: lager (a light roasted beer) and stout (a dark roasted beer) with those of an equivalent amount of pure ethanol on hepatic CYP expression. Beer or pure ethanol was part of a complete total enteral nutrition diet that was infused intragastrically into male Sprague Dawley rats for 21 days. At the end of the infusion period, rats were euthanized, and liver and intestinal microsomes were prepared. Expression and activity of CYP1A1/2, CYP2B1, CYP2E1, CYP3A, and CYP4A were assessed by Western immunoblotting and by using CYP enzyme-specific substrates, respectively. RESULTS: mRNA and protein levels of CYP4A1 were elevated only in stout-treated animals. However, lauric acid 12-hydroxylase activity (a CYP4A-specific activity) was reduced (p < or = 0.05) in microsomes from lager- and stout-fed rats. After oxidation with potassium ferricyanide, this activity was significantly increased in microsomes from stout-fed animals. The relative expression of CYP2E1 and CYP2B1 and the activities toward p-nitrophenol, pentoxyresorufin, or benzyloxyresorufin did not differ between beers or compared with pure ethanol or controls. However, the mean expression of CYP1A2, CYP3A, and CYP4A apoproteins was greater in liver microsomes from stout-infused rats than in those from lager-infused rats, ethanol-infused rats, and diet controls (p < or = 0.05). In addition, although no significant differences were observed in ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), midazolam, or testosterone hydroxylase activities between groups, stout-infused rats had greater hepatic microsomal erythromycin N-demethylase activity than other groups (p < or = 0.05). CONCLUSIONS: Stout contains components other than ethanol that interact in a complex fashion with the monooxygenase system.     

Granuloma collagenase and EDTA-sensitive neutral protease production in hepatic murine schistosomiasis

Mice infected with Schistosoma mansoni represent a model for study of hepatic fibrosis in humans. Production of trypsin-activatable inactive collagenase and EDTA-sensitive neutral protease was measured in the culture medium in which granuloma explants or primary cultures were maintained. Collagenase production was maximal in granulomas obtained from liver of mice 8 weeks postinfection and was inhibited by Actinomycin D or cycloheximide, and enhanced by lymphocyte factor(s) or heparin. Isolated schistosome eggs did not release these enzymatic activities but did release EDTA-insensitive protease activity. Both enzymes were separated by ion-exchange chromatography and purified to homogeneity. Isolated collagenase had an isoelectric point of 6.2 and molecular weight of 60,000 and had the functional characteristics of a tissue collagenase. The specific activity of collagenase was 33 units per mg protein at an optimum pH 7.5 and lacked proteolytic activity against noncollagenous protein substrates. Isolated EDTA-sensitive neutral protease had specific caseinolytic activity of 150 units per mg protein and gelatinolytic activity of 300 units per mg protein at an optimum pH 7.5; the enzyme lacked activity against undenatured collagen. Isoelectric point was pH 6.0. Protease activity was inhibited by known inhibitors of collagenases. Production and activation of EDTA-sensitive neutral protease and collagenase accompany increased collagen synthesis and content in the liver of mice 8 weeks postinfection with S. mansoni cercariae. Continued accumulation of liver collagen under these conditions suggests an insufficiency in collagenase activity relative to the increase in collagen synthesis.