Impact of lipid and ACE inhibitor therapy on cardiovascular disease and metabolic abnormalities in the diabetic and hypertensive patient

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2alpha-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was < 10%. Furthermore, immunoblotting showed that 1,1-DCE (> or = 400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6beta-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40-70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid omega-hydroxylase (LAOH), and testosterone 7alpha-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60-66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (approximately 1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE.     

Changes in rat liver cytochrome P450 enzymes by atrazine and simazine treatment

1. We examined the effect of two chloro-s-triazines (atrazine and simazine) on hepatic microsomal cytochrome P450 enzymes in rat. Rats were treated intraperitoneally with atrazine or simazine daily for 3 days with 100, 200 and 400 mumol/kg. 2. Among the P450-dependent monooxygenase activities, testosterone 2 alpha-hydroxylase (T2AH) activity in rat, which is associated with CYP2C11, was significantly decreased at all doses of atrazine and simazine. The levels relative to control activities were 59-46 and 60-32% respectively. Similarly, oestradiol 2-hydroxylase (ED2H) activity was also significantly decreased by 28-51% by atrazine and simazine at all doses. However, no change in CYP2C11 protein level by either chloro-s-triazine was observed. K(m) for T2AH was significantly increased only by simazine (200 mumol/kg), whereas the Vmax and Cl(int) for T2AH were significantly decreased by atrazine and simazine at all doses. 3. 7-Ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and 7-pentoxyresorufin O-depentylase (PROD) activities were significantly increased by 1.4-1.6-, 1.7-3.2- and 1.5-2.2-fold respectively, by both chloro-s-triazines at 200 or 400 mumol/kg. Lauric acid omega-hydroxylase (LAOH) was also increased by 1.4-fold by simazine at 200 and 400 mumol/kg. Immunoblotting showed that only simazine induces CYP1A2 and CYP4A1/2 protein expression. 4. The activities of 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1'-hydroxylase (BF1'H), chlorzoxazone 6-hydroxylase (CZ6H), testosterone 6 beta-hydroxylase (T6BH) and testosterone 7 alpha-hydroxylase (T7AH) were not affected by either chloro-s-triazine. 5. These results suggest that the pattern of changes in P450 isoforms by chloro-s-triazines differs between atrazine and simazine, that these herbicides change the constitutive and/or male specific P450 isoform(s) in rat liver, and that these changes closely relate to the toxicity of chloro-s-triazines.     

Induced abortion as an independent risk factor for breast cancer: a comprehensive review and meta-analysis

Effects of continuous feeding flavonoids (flavone, flavanone, and tangeretin) on drug-metabolizing enzymes in rat liver were investigated to ascertain how long feeding is required to reach maximal induction and to determine whether maximal induction is maintained for a long period of feeding. In the first experiment rats received a diet containing 10 mmol flavonoid/kg dry matter for 4, 8, 16, or 32 d. The second experiment was designed to examine the time course for induction during the first 4 d. The kinetics of induction depended on the chemical structure of the flavonoid and was different from one enzyme to another. Flavone increased P450 1A and P450 2B apoproteins and stimulated many enzyme activities. A significant increase of P450 1A1/2 proteins, ethoxyresorufin O-deethylase (EROD), and methoxyresorufin O-demethylase (MROD) activities occurred as early as 6 h after the first administration, and a gradual increase was observed up to 4 d of feeding. P450 2B1/2 proteins and pentoxyresorufin O-depentylase (PROD) activity were also increased but after a lag period when compared with P450 1A1/2 proteins. EROD and MROD activities declined after 4 d, whereas PROD activity remained steady during 32 d of flavone feeding. Glutathione transferase (GST) and p-nitrophenol UDP-glucuronosyl transferase (UGT) activities were also increased. The maximal induction was reached by 4 d of feeding for UGT and after a longer duration of feeding (16 d) for GST. Flavanone treatment induced mostly P450 2B1/2 proteins and PROD, GST, and UGT activites. After 4 d of feeding, P450 2B1/2 proteins and PROD activity declined whereas GST and UGT activities remained steady. Tangeretin treatment produced changes similar to flavone but of lesser magnitude and after a longer delay.     

Inducibility of enzyme activities associated with the cytochrome P-450 1A family, ethoxyresorufin O-deethylase, and methoxyresorufin O-demethylase in human hepatocyte lines derived from normal liver tissue

Three human hepatocyte cultures have been developed from specimens of normal human liver, in each case from an infant or child, by coculture with liver epithelial cells from 6-day-old rat pups in a complex growth medium. In the established cultures hepatocytes predominate and maintain typical hepatocellular morphology by light microscopy and albumin secretion into supernatant medium. The activity of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) basally and after treatment with polycyclic aromatic hydrocarbons was measured spectrofluorometrically in cell homogenates from each culture. Very low levels of EROD and MROD activity were found in each culture without induction [EROD: 1.78 +/- 0.71 (mean +/- SE) pmol/min/mg protein; MROD: 1.33 +/- 0.10 pmol/min/mg protein]. After treatment with 10 microM dibenz (a,h)anthracene x 48 hr, EROD and MROD activities rose approximately 20- to 50-fold. When the basal and induced enzyme activities were remeasured 2 months later, results were essentially the same. Incubation with 10 microM benz(a)anthracene x 48 hr also led to induction of EROD and MROD activities. We believe that these cultures can be regarded as human hepatocyte lines, which conserve human hepatic polycyclic aromatic hydrocarbon-inducible P-450s, most likely including P-450 1A2.     

Crystal structure of the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability

The in vivo and in vitro effects of the insecticide deltamethrin (DM) on hepatic cytochrome P450 (Cyt P450) monooxygenase were examined in adult carp. The in vivo experiments were carried out with 0.2 microgram/l DM at 20 degrees C. The changes in the hepatic microsomal Cyt P450 content and the Cyt P450-dependent monooxygenase activities were studied in DM-treated fish. Although there were no changes in the Cyt P450 content during the exposure time, after treatment for 24 h all the investigated isoenzyme activities (para-nitrophenetole-O-deethylase, p-NPOD; aminopyrene-N-demethylase, APND; ethylmorphine-N-demethylase, EMND; 7-ethoxycoumarin-O-deethylase, ECOD; and ethoxyresorufin-O-deethylase, EROD) were significantly inhibited. After 72 h, all the activities were still lower than in the control animals. In vitro incubation of liver microsomes with DM led to a concentration-dependent decrease in total microsomal Cyt P450 content. A complete loss of Cyt P450 occurred after a 5-min incubation with 60 microM DM. The maximum in the difference spectra of microsomes was shifted to higher wavelength, showing the strong interaction of DM with Cyt P450. EROD and ECOD activities were inhibited by DM. The in vitro kinetic results on ECOD revealed that the inhibition was of non-competitive type, with K1 = 9.8 +/- 2.3 microM. This study indicates important biochemical effects of DM in fish liver, and suggests that exposure to DM may cause loss of the Cyt P450-dependent metabolism in fish.     

Induction and inhibition of cytochrome P450 isoforms by imazalil, a food contaminant, in mouse small intestine and liver

1. The effects of imazalil, a food contaminant used as a fungicide, were investigated on the expression and activity of cytochrome P450 in the small intestinal mucosa and liver of mice. Imazalil was orally administered to mice daily at 1 or 10 mg/kg for 3 days. 2. Imazalil enhanced cytochrome P450-catalysed ethoxyresorufin O-deethylase and pentoxyresorufin O-depentylase (PROD) activities in both tissue microsomes at the 10 mg/kg/day dose level, indicating the induction of cytochrome P450 subfamilies CYP1A and CYP2B. In addition, immunochemical analyses also demonstrated an enhanced expression of CYP2B, CYP2C and CYP3A subfamilies in both tissues. 3. Imazalil was a potent inhibitor of cytochrome P450-dependent monooxygenase activities (PROD, aminopyrine N-demethylase and erythromycin demethylase) in in vitro assays using both small intestinal and liver microsomes. 4. From these findings, imazalil has been demonstrated to have not only a potent inhibitory activity but also a significant inducing ability of P450 isoforms in the small intestine. Prolonged ingestion of such a food contaminant may modulate the xenobiotic-metabolizing enzyme system at the site of a primary portal of xenobiotic entry to the systemic circulation.     

Inhibition of murine hepatic cytochrome P450 activities by natural and synthetic phenolic compounds

1. The effect of the phenolic compounds protocatechuic acid, chlorogenic acid, tannic acid, gallates and silybin on ethoxyresorufin O-dealkylase (CYP1A1), methoxyresorufin O-dealkylase (CYP1A2) and pentoxy-O-dealkylase (CYP2B) was examined in mouse liver microsomes from induced animals. 2. All compounds tested could inhibit cytochrome P450-mediated enzyme activities, but to different extents. Tannic acid was the most potent inhibitor, especially toward EROD activity with an IC50=2.6 microM. Synthetic dodecyl gallate was also relatively selective toward this enzyme activity with an IC50=120 microM. 3. Protocatechuic acid, chlorogenic and silybin were more selective towards PROD and MROD activities. Their relative inhibitory potency for PROD activity was as follows: chlorogenic acid > protocatechuic acid > silybin > dodecyl gallate > propyl gallate. Protocatechuic acid was a more effective inhibitor of MROD activity than chlorogenic acid, and propyl gallate more effective than dodecyl gallate. Thus, no clear structure-activity or selectivity relationship was observed. 4. Analysis of the kinetics of inhibition revealed that the inhibition in most cases was non-competitive in nature.     

Time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic drug-metabolizing enzyme activity in rats

The time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic cytochrome P450-dependent drug-metabolizing capacity (cytochrome P450 and b5 content, activity of aminopyrine N-demethylase, p-nitroanisole O-demethylase, aniline hydroxylase and benzphetamine N-demethylase) and on the pharmacokinetics of antipyrine have been determined in rats. Measurement of enzyme activity and antipyrine (after intravenous injection of 20 mg kg(-1)) were performed 2, 24 and 96 h after a single intraperitoneal injection of endotoxin (1 mg kg(-1)) and after repeated doses (once daily for 4 days). The contribution of tumour necrosis factor alpha (TNFalpha) to the endotoxin-induced changes was also examined in rats pretreated with granulocyte colony-stimulating factor (G-CSF). The systemic clearance of antipyrine and the activity of hepatic cytochrome P450-dependent drug-metabolizing enzymes were dramatically reduced 24 h after a single injection of endotoxin, but had returned to control levels by 96h. The magnitudes of these decreases in these measurements after repeated doses of endotoxin were similar to those seen 24h after the single dose. The systemic clearance of antipyrine correlated significantly with cytochrome P450 content and aminopyrine N-demethylase activity. In histopathological experiments, moderate hypertrophy of Kupffer cells was observed, with no evidence of severe liver-tissue damage. G-CSF pretreatment suppressed the increased plasma concentrations of TNFalpha produced 2 h after single endotoxin injection, but did not eliminate the endotoxin-induced decrease in the systemic clearance of antipyrine, suggesting that TNFalpha is not the sole component responsible for the reduction of cytochrome P450-mediated drug-metabolizing enzyme activity. These results provide evidence that a single intraperitoneal injection of 1.0 mgkg(-1)K. pneumoniae endotoxin in rats reduces hepatic P450 and b5 levels, and reduces the activity of various cytochrome P450-mediated drug-metabolizing enzymes without causing severe liver-tissue damage. This suggests that the effect of endotoxin on hepatic cytochrome P450-mediated drug-metabolizing isozymes is non-selective.     

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon beta in mice

AIMS/METHODS: Interferon beta is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon beta on cytochrome P-450 in mice. RESULTS: Interferon beta (2 x 10(5) units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon beta reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In beta-naphthoflavone-treated mice, interferon beta lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). CONCLUSIONS: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon beta, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon beta.     

Methoxyresorufin: an inappropriate substrate for CYP1A2 in the mouse

Hepatic microsomes derived from Cypla2(-/-) knockout (KO) and parental strains of mice, C57BL/6N and 129Sv, were used to examine the specificity of methoxyresorufin and acetanilide as substrates for CYP1A2 activity. In addition, animals from each group were exposed to CYP1-inducing compounds. As expected, microsomes from untreated 1a2 KO mice did not have immunodetectable CYP1A2 protein; however, methoxyresorufin-O-demethylase (MROD, 25.5+/-6.1 pmol/min/mg protein) and acetanilide-4-hydroxylation (ACOH, 0.64+/-0.04 nmol/min/mg protein) activities were still present. Furthermore, induction of ethoxyresorufin-O-deethylase (EROD) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 1a2 KO mice was accompanied by a greater than 70-fold increase in MROD activity. In contrast, ACOH was only induced 2-fold by TCDD. As with 1a2 KO mice, the parental strains exposed to TCDD or 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) showed substantial EROD and MROD induction, whereas ACOH activity was induced to a lesser degree. PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) resulted in low levels of both EROD and MROD induction. Results indicate that both substrates are subject to metabolism by non-CYP1A2 sources, and the apparent contribution of CYP1A1 activity to methoxyresorufin metabolism makes MROD unsuitable for differentiating CYP1A1 and CYP1A2 activities in the mouse.     

Investigation on possible antioxidative properties of the NMDA-receptor antagonists ketamine, memantine, and amantadine in comparison to nicanartine in vitro

Possible antioxidative properties of three N-methyl-D-aspartate (NMDA)-receptor antagonists, the anesthetic ketamine and the antiparkinson drugs memantine and amantadine were investigated in vitro on the microsomal cytochrome P450 (P450) system of rat livers and on rat whole blood chemiluminescence in comparison to nicanartine, a substance with known antiatherosclerotic, hypolipemic and antioxidative capacity. For this purpose, the effects on NADPH- and iron-stimulated lipid peroxidation (LPO), hydrogen peroxide (H2O2) production, and NADPH- and iron-stimulated lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) were examined using rat liver microsomes. Additionally, the influence on LM amplified whole blood chemiluminescence after zymosan activation of polymorphonuclear leukocytes (WB-CL) was investigated. Furthermore, binding to P450 and effects on P450 mediated monooxygenase function, as measured by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (END), were assessed. Nicanartine concentration dependently reduced LPO and H2O2 production already at a concentration of 1 microM, whereas LC and LM amplified CL and WB-CL were not affected. EROD and END were concentration dependently diminished starting at 1 microM, and ECOD already at 0.1 microM. Ketamine decreased LPO, H2O2 production and LM and LC amplified CL, starting at 100 microM. WB-CL was significantly diminished already at 10 microM. EROD and ECOD were inhibited at 10 and 100 microM and END at 100 microM. With memantine a concentration dependent inhibition of LPO and WB-CL was seen at 100 and 1000 microM and a reduction of LC and LM amplified CL only at 1000 microM. H2O2 production was not affected. EROD and ECOD were significantly diminished by a concentration of 100 microM. No effect was observed on END. Amantadine significantly reduced LPO and WB-CL, but only at 1000 microM. H2O2 production and LC and LM amplified CL were not affected. EROD was significantly diminished at 100 microM, whereas no influence was seen on ECOD and END. Nicanartine displayed type II or reverse type I, ketamine, memantine and amantadine type I substrate binding to P450. The highest binding affinity to P450 was seen with nicanartine, followed by ketamine, memantine and then amantadine. These results demonstrate, that all four substances seem to act as radical scavengers and/or as inhibitors of the oxidative function of P450. All four substances seem to interfere with the monooxygenase function of P450. This may result in a possible influence on the biotransformation of endogenous as well as of foreign compounds. The effects of nicanartine were much more pronounced than those of ketamine, memantine, and amantadine.     

Drug metabolism in hepatocyte sandwich cultures of rats and humans

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 micromol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.     

Tissue distribution, toxicokinetics and induction of hepatic drug metabolizing enzymes in male rats after a single s.c. dose of 3,4,3',4'-tetrachlorobiphenyl (PCB-77)

The tissue distribution and toxicokinetics of PCB-77 after a single s.c. dose of 6 mg/kg body weight was investigated in male adult rats as well as the concurrent induction of hepatic drug-metabolizing enzymes. During five days after treatment, the PCB-77 concentrations on fat weight basis in testis and in whole blood were found to be about in the same range as the concentrations in adipose tissue, whereas in liver fat PCB-77 accumulated. EROD and MROD activities were induced by about 8 and 4 times, respectively, during a period of two weeks after treatment. Concentrations in adipose tissue and in liver as well as the hepatic enzyme activities rapidly declined during the investigation period of eight weeks. An elimination half-life for PCB-77 of approximately 7-9 days was estimated.     

Interactions of the bacterial pathogen Listeria monocytogenes with mammalian cells: bacterial factors, cellular ligands, and signaling

Risedronate ([1-hydroxy-2-(3-pyridinyl)-ethylidene[bis]phosphonic acid] monosodium salt) was evaluated for induction of hepatic microsomal drug metabolizing enzymes in male and female Sprague Dawley rats (N = 4/sex/dose group). Main study animals received water (vehicle control), risedronate (0.1, 0.8, 4, or 16 mg/kg/day) or phenobarbital (80 mg/kg/day, positive control) by daily oral gavage for 14 consecutive days. Recovery study animals received water, risedronate (16 mg/kg/day) or phenobarbital (80 mg/kg/day) by daily oral gavage for 14 consecutive days and then were maintained drug-free for 14 days to evaluate the reversibility of any observed effects. At the conclusion of each study the animals were sacrificed, the liver removed, weighed and the microsomal subcellular fraction prepared. The hepatic microsomal fraction was then evaluated for protein content, cytochrome P450, and the activities of aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase. Risedronate was well tolerated during the dosing phase of the study as evidenced by clinical observations, body weight gain and food consumption which were not significantly different from the vehicle controls. Risedronate did not significantly increase (P > 0.05) liver weight, liver/body weight ratio, protein content, P450, aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase or p-nitrophenol UDP-glucuronosyltransferase in rats of either sex when compared to vehicle controls. As expected, the hepatic microsomal enzyme inducer phenobarbital significantly increased (P < 0.05) liver weight, liver/body weight ratio, protein content (males only), P450, aniline hydroxylase (males only), aminopyrine N-demethylase (males only), ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase in rats relative to vehicle controls. Following the 14 day drug-free recovery period the induction parameters increased by phenobarbital reversed to vehicle control levels. The results obtained in this well controlled study indicate that risedronate is not an inducer of hepatic microsomal drug metabolizing enzymes in the rat.     

Interaction of nonylphenol and hepatic CYP1A in rats

Both estradiol and nonylphenol (NP) inhibited hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of beta-naphthoflavone-treated rats. Enzyme kinetic analyses (Lineweaver-Burk plots) using different estradiol and NP concentrations with graded increases in the concentrations of the substrate, ethoxyresorufin, showed that the inhibition was of a competitive nature at all concentrations of estradiol or NP used. Thus, the mechanism by which NP inhibits EROD activity is similar to that of estradiol. NP, however, was much less potent than estradiol. Young rats treated in vivo with 80 mg/kg body weight of NP demonstrated a slight but significant decrease in their hepatic microsomal EROD activity and CYP1A protein as measured by western blot analysis. In addition, treatment with NP led to a decrease in the steady-state levels of hepatic CYP1A mRNA in rats, suggesting that NP acted at the pre-translational level. The competitive nature of inhibition by NP on hepatic microsomal EROD activity indirectly suggests that this compound is a possible substrate of the CYP1A enzyme. Furthermore, NP had a moderate modulating effect on the expression of CYP1A in rat liver.     

Effects of triphenyltin and other organotins on hepatic monooxygenase system in fish

The interaction of the organotin fungicide triphenyltin chloride (TPT) with fish microsomal monooxygenase systems has been studied in vitro and in vivo in the marine fish scup (Stenotomus chrysops). In vitro incubation of fish liver microsomes with TPT resulted in the conversion of about 40% of the native total spectral P450 to P420. In addition, a strong concentration-related inhibition of ethoxyresorufin O-deethylase (EROD) activity was observed, with a complete loss at 1.0 mM TPT. Pentoxyresorufin-O-dealkylase (PROD) activity was inhibited only at the highest concentration tested. This suggests either some specificity for the EROD catalyst CYP1A1, or a loss of reductant NADPH cytochrome c reductase as the cause. Further in vitro incubations showed that NADPH, but not NADH, cytochrome c reductase was strongly inhibited at 100 microM TPT and higher. To further investigate this effect, fish were injected with single doses of 5, 25 and 50 microM TPT (1.9, 9.6 and 19.3 mg kg-1 TPT), and 24 and 48 h later, hepatic microsomes were analyzed for total P450 content, EROD activity, NAD(P)H cytochrome c reductase, and the content of three CYP forms. EROD activity tended to be decreased in TPT-treated scup, with the response being stronger after 48 than 24 h. No significant conversion of spectrally determined P450 to cytochrome P420 was found, and cytochrome b5 was not affected. However, both NAD(P)H cytochrome c reductases were significantly inhibited at all concentrations. Immunoblot analysis showed reduction of CYP1A1 content at all doses, being significant at 25 mM after 48 h, but no decrease in CYP3A-like protein, the dominant catalyst of testosterone 6 beta-hydroxylation, nor CYP2B-like protein, the major contributor to indicates significant effects of TPT at high concentrations on fish hepatic CYP1A1 protein, EROD activity and the reductases. TPT seems to act more specifically on CYP1A1 than on other CYP forms. These findings combined with those of our previous studies (Brüschweiler BJ, Würgler FE, Fent K. Environ Toxicol Chem 1996;15:827-735; Fent K, Bucheli TD. Aquat Toxicol 1994;28:107-126; Fent K, Stegeman JJ. Aquat Toxicol 1991;20:159-168; Fent K, Stegeman JJ. Aquat Toxicol 1993;24:219-240) indicate a general degenerative effect of organotins on the fish microsomal monooxygenase system, although some differences are seen between the organotins, and between species. We conclude that these effects of organotins have consequences for use of CYP1A as a biomarker and endocrine disruption.     

Time course of enzyme induction in liver and kidneys and absorption, distribution and elimination of 1,4-dichlorobenzene in rats

Time course of enzyme induction was measured in Fischer344 rats treated daily at 150 and 600 mg 1,4-dichlorobenzene (1.4-DCB)/kg p.o. up to 28 days. The monoxygenases 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and aldrin epoxidase (ALD) as well as the phase II enzymes; epoxide hydrolase (EH), glutathione S-transferase (GS-T) and glucuronyl transferase (GLU-T) were dose-dependently induced in the liver of males and females. A pronounced induction in the kidneys was measured at 600 mg/kg only for ECOD. After single oral administration of 100 and 1000 mg/kg bw and feeding of 100 and 1000 ppm (corresponding to approximately 10 and 100 mg/kg bw) to male Wistar rats for 28 days, the time course of 1,4-DCB and 2,5-DCP concentrations was investigated in plasma, adipose, hepatic and renal tissue. In addition, total urinary excretion of 2,5-DCP was determined. After single application, 1,4-DCB and 2,5-DCP were rapidly eliminated from the plasma and tissues, 40-60% of the dose administered was excreted as 2,5-DCP in the urine. There were no indications of cumulative effects after a feeding period of 28 days. The concentrations decreased in all tissues until the 7th day of study. Thereafter, there seems to be a steady state until the 28th day. A total of 7 days after the end of exposure, no more residues could be detected. Following long-term inhalation (450 and 3000 mg/m3) 1,4-DCB concentrations were highest in adipose tissues at 6 months followed by a marked decline at 18 months. 1,4-DCB and 2,5-DCP concentrations in plasma and liver were much lower but again with a peak at 6 months. When compared with published human data on measurements in plasma, urine, liver and adipose tissue the results suggest that there should be no hazard for the general population.     

Induction of cytochrome P450 1A1 in rat liver slices by 7-ethoxycoumarin and 4-methyl-7-ethoxycoumarin

7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.