Cytochrome P450-dependent Monooxygenase Activities Research Articles

Effect of 4-tert-octylphenol on cytochrome P450 enzymes in rat liver.

The effects were studied of 4-tert-octylphenol (OP) on hepatic cytochrome P450 enzymes in rats. Rats were treated intraperitoneally with OP twice, at doses of 5, 10, and 20 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, testosterone 2alpha-hydroxylase activity, which is associated with CYP2C11, was significantly decreased by OP at all doses. The level relative to control activity was 67-22%. CYP3A2-dependent monooxygenase, testosterone 6beta-hydroxylase activity was also decreased by 51% by OP at 20 mg/kg. Furthermore, immunoblotting showed that OP (10 or 20 mg/kg) significantly decreased CYP2C11/6 and CYP3A2/1 protein levels. However, the reduction ratio of CYP2C11/6 and CYP3A2/1 protein levels by OP treatment was lower than that of testosterone 2alpha-hydroxylase and testosterone 6beta-hydroxylase activities. The Cl(int) (Vmax/Km) value for testosterone 2alpha-hydroxylase was significantly decreased by OP at all doses, whereas the Cl(int) value for testosterone 6beta-hydroxylase was only decreased by OP at 20 mg/kg. In addition, 7-ethoxycoumarin O-deethylase activity was significantly decreased by 32% by the highest dose of OP. By contrast, CYP1A1-, CYP1A2-, CYP2A1-, CYP2B1/2-, CYP2D1-, CYP2E1- and CYP4A1/2/3-dependent monooxygenase activities were not affected by OP at any dose. These results suggest that OP changes the male-specific cytochrome P450 isoforms in rat liver, and that these changes closely relate to the toxicity of OP.

Assessment of the Bioavailability of PAHs in Rats Exposed to a Polluted Soil by Natural Routes: Induction of EROD Activity and DNA Adducts and PAH Burden in Both Liver and Lung

In order to assess bioavailability of polycyclic aromatic hydrocarbons (PAHs) present in soils, male laboratory rats were exposed to litters of control and polluted soils. After 88±2 h of exposure, several biomarkers were measured in both liver and lung. When rats were exposed to SIV soil, contaminated by a mixture of at least 13 PAHs, (1) only 2 or 3 PAH compounds were detected in liver and lung; (2) cytochrome P450-dependent monooxygenase activity, followed by 7-ethoxyresorufin O-deethylase (EROD) activity measurement, was highly induced in liver (13-fold-induction) and lung (up to 78-fold); and (3) DNA adducts were significantly increased. For what concerns soil artificially contaminated by only one PAH (phenanthrene or B[a]P), EROD activity was not or fewly induced, respectively. These results demonstrate the occurrence of a high bioavailability of PAHs to mammals in natural conditions of exposure. First results concerning DNA adducts must be profounded, but they already show that a short exposure of mammals to PAH-polluted soils can lead to potential genotoxic effects. EROD activity can be used as a sensitive biomarker in both liver and lung of rats maintained on litters of soils in the laboratory, and such a test can be used routinely to contribute to risk assessment.

Altered Cholinesterase and Monooxygenase Levels inDaphnia magnaandChironomus ripariusExposed to Environmental Pollutants

Biochemical indices were investigated for their potential use as variables of sublethal toxicity inDaphnia(cholinesterase) andChironomus(cholinesterase and biotransformation enzymes). Parathion, dichlorvos, and aldicarb caused dose-related inhibition of cholinesterase (ChE) in 24-h bioassays with both species. Ratios ofDaphniaandChironomusChE IC50values to corresponding immotility EC50values derived from the same experiment covered the range 0.26 to 1.2. Estimates of the ChE inhibition caused by the immotility EC50were in the range 53–99% below control activity. ChE IC50values of dichlorvos, parathion, and aldicarb were 0.17, 0.61, and 95 μg/liter inDaphniaand 6.2, 2.9, and 27 μg/liter inChironomus, respectively. Cytochrome P450-dependent monooxygenase activities (ethoxyresorufin-O-deethylase, methoxyresorufin-O-deethylase, and ethoxycoumarin-O-deethylase) were detectable inChironomusbut not inDaphnia.Chironomusmonooxygenase activities were significantly inhibited to about 30% of control values after 4 days of exposure to 50 μg/liter 3,4-dichloroaniline but remained unchanged by 0.5 μg/liter parathion. An approximately 1.3-fold induction of monooxygenase activities was caused by the model inducer naphthalene (0.1mg/liter). These results suggest that cytochrome P450-dependent monooxygenase activities may be useful variables in toxicity tests with aquatic insects.

Bioavailability of PCBs to male laboratory rats maintained on litters of contaminated soils: PCB burden and induction of alkoxyresorufin O-dealkylase activities in liver and lung.

Male rats from the Sprague-Dawley laboratory strain were maintained in the laboratory during 3 days and 1 night on litters containing a reference soil and different amounts of a soil, mainly polluted by PCBs (207 ppm expressed in Aroclor(R) 1254; SIII soil). Two categories of biomarkers of exposure were measured in both liver and lung of these rats: PCB burdens and activities of microsomal liver and lung cytochrome P450-dependent mono-oxygenases, namely ethoxy-, pentoxy-, and benzoxy-resorufin O-dealkylase activities (EROD, PROD, and BROD, respectively). PCB burdens in liver and lung of rats exposed to SIII soil were 1,845 and 241 ppb, respectively (expressed in Aroclor(R) 1254 equivalents). EROD, PROD, and BROD were significantly induced in the liver of rats exposed to SIII soil, while only EROD activity was induced in the lung. Induction of hepatic EROD activity was approximately 3- to 5.4-fold; pulmonary EROD activity was induced by 9- to 12-fold. In the lung, PROD and BROD activities were inhibited. When rats were exposed to SIII soil diluted with various amounts of standard ISO soil, a nearly linear dose-response relationship was found between the level of PCBs in the litter and EROD activity in both liver and lung. A nonlinear dose-response relationship exists with hepatic BROD activity; no dose-response relationship was observed with hepatic PROD and pulmonary PROD and BROD activities. EROD activity measurement in both liver and lung of rats maintained on a litter of PCB polluted soil was used to assess the bioavailability to mammals of PCBs.

Carbon monoxide as a regulator of bile canalicular contractility in cultured rat hepatocytes.

This study aimed to examine the mechanism(s) by which carbon monoxide (CO), a product of heme oxygenase reaction, controls the contractility of bile canaliculus (BC) in hepatocytes. When BCs associated with the couplet cells in cultured rat hepatocyte suspension were observed using time-lapse video microscopy, they exhibited periodical contractions with a most-probable interval of 6 minutes under our experimental conditions. The addition of 1 micromol/L zinc protoporphyrin IX (ZnPP), a potent inhibitor of heme oxygenase, to the culture medium elicited a 40% shortening of the interval time together with an increase in intracellular calcium concentrations, while the same concentration of iron protoporphyrin IX did not induce such changes. The production of CO, which was 0.5 nmol/h/10(8) cells in the absence of ZnPP, diminished to less than 0.1 nmol/h/10(8) cells upon application of ZnPP. The ZnPP-elicited increases in both contractile frequency and intracellular calcium concentrations were attenuated by the addition of 1 micromol/L CO or 50 micromol/L 1,2-bis(2-aminophenoxy) ethane-tetraacetate, a calcium chelator. Clotrimazole or metyrapone, inhibitors of cytochrome P450-dependent monooxygenase activities, also attenuated the ZnPP-induced elevation of the contractile frequency. On the other hand, intracellular cyclic guanosine monophosphate (cGMP) contents were not altered significantly by the application of ZnPP or by CO. These results indicate that CO generated by heme oxygenase controls the BC function by changing intracellular calcium concentrations presumably through a mechanism involving the cytochrome P450 reaction.

Sulfotransferase-mediated genotoxicity of propane 2-nitronate in cultured ovine seminal vesicle cells

2-Nitropropane (2-NP) is a well-known genotoxin and carcinogen in rat liver. Several metabolic pathways, particularly cytochrome P450-, peroxidase- and sulfotransferase-dependent ones, have been suggested to lead to the formation of DNA-reactive species from 2-NP. Because rat liver cells express most types of xenobiotic-metabolizing enzymes, the role of specific pathways in the metabolic activation of 2-NP is difficult to assess in these cells. We have therefore investigated the genotoxicity of 2-NP and its anionic form, propane 2-nitronate (P2N), in cultured ovine seminal vesicle (OSV) cells. OSV cells lack cytochrome P450-dependent monooxygenase activity, but express prostaglandin- H-synthase (PHS) and, as we found out, phenol sulfotransferase. The induction of DNA repair synthesis and specific DNA modifications served as indicators for the genotoxicity of 2-NP and P2N. Both forms strongly induced repair, P2N being more active than 2-NP. The secondary nitroalkanes nitrocyclopentane and nitrocyclohexane also induced repair, whereas 1-nitropropane and the reduction product of 2-NP, acetone oxime, did not. P2N also elicited the formation of the characteristic DNA modifications `DX1' and 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine residues in the DNA. Pretreatment of OSV cells with indomethacin, an inhibitor of PHS, affected neither the induction of repair nor the formation of the DNA modifications, and P2N was not a reducing substrate for the PHS-peroxidase activity. In contrast, the sulfotransferase inhibitor pentachlorophenol strongly reduced genotoxicity. The results show that cytochrome P450-dependent monooxygenases are not required for the metabolic conversion of secondary nitroalkanes or their nitronates into DNA-damaging products, nor is PHS involved in the metabolic activation. Instead, the data corroborate an essential role of sulfotransferase(s) in the genotoxicity and carcinogenicity of secondary nitroalkanes. Moreover, it is demonstrated for the first time that these compounds can be genotoxic in cells other than hepatocytes or hepatoma cells. This implies that in species other than the rat, organs other than the liver can be targets for the genotoxicity, and possibly carcinogenicity, of secondary nitroalkanes.

Acute and subacute toxicity study of water-soluble polyalkylsulfonated C60 in rats.

Polyalkylsulfonated C60, or FC4S, a highly water-soluble caged fullerene derivative, is believed to be a free radical remover or an antioxidant in biological systems. A 50 mg/ml aqueous solution was prepared as a master solution and administered to female Sprague-Dawley CD(Crl:CD(SD)BR) rats in a single-dose acute toxicity study or a 12-day subacute toxicity study where rats were given the solution daily. In a study of the median lethal dose (LD50), no rats died after oral administration, and thus FC4S was considered to be nontoxic if administered orally. In an LD50 intraperitoneal injection study, rats died within 30 hr after injection; the LD50 was determined to be approximately 600 mg per kilogram of body weight. Rats injected with the compound intraperitoneally or intravenously immediately eliminated the compound through the kidney; the kidney appeared to be the primary target organ. The compound induced a distinct lysosome-overload nephrosis, a phagolysosomal nephropathy characterized by a tinctorial difference between the outer cortex and the inner cortex and the medulla. The affected outer cortex showed a diffuse degeneration, with the presence of numerous large vacuoles and cytoplasmic aggregates in the tubular epithelium. The phagolysosomal nephropathy was detected in rats after acute exposure as well as in the surviving rats following 1 intraperitoneal injection of 500 mg/kg or intravenous injection of 100 mg/kg. Ultrastructural investigation revealed numerous membranous conglomerates characteristic of phagolysosomal and/or lysosomal inclusions in the cytoplasm of the renal tubular epithelium. These conglomerates were confined to the vacuole, electron-dense, and unevenly stained. They varied in size and shape and were fused or aggregated. Occasional phagolysosomes were also observed in the endothelial cells of the peritubular plexus. A preliminary study of microsomal enzyme activity analysis revealed a suppression effect of liver cytochrome P-450-dependent monooxygenase activities, including cytochrome P-450, cytochrome b5, and benzo(a)pyrene hydroxylase, but an increased level of kidney cytochrome P-450-dependent monooxygenase activities, including NADPH-cytochrome P-450 reductase. The significance of these enzyme alterations was not well determined. Further study is needed to clarify the correlation between the alterations of microsomal enzyme activity and the nephropathy of lysosomal overload-induced changes. These changes may serve as a biological marker in toxicity screening tests for this class of compound.

Monooxygenase activities in carp as biochemical markers of pollution by polycyclic and polyhalogenated aromatic hydrocarbons: choice of substrates and effects of temperature, gender and capture stress

Cytochrome P450-dependent monooxygenase activities (7-ethoxyresorufin, 7-pentoxyresorufin and 7-ethoxycoumarin O-dealkylases and aminopyrine N-demethylase) were measured in hepatopancreatic microsomes of carp collected in one control and one polychlorinated biphenyl- and polycyclic aromatic hydrocarbon-polluted ponds in the Czech Republic. The magnitudes of responses of monooxygenase activities to the exposure to polycyclic and polyhalogenated aromatic compounds (PAC), namely polycyclic aromatic hydrocarbons and polychlorinated biphenyls, and effects of other factors including ambient temperature and stress associated with reduction of water volume at capture were assessed. An increase of the level of 7-ethoxyresorufin O-deethylase (EROD) i.e. the specific monooxygenase activity mediated by the cytochrome P4501A (CYP1A) isoenzymes, was the most sensitive biochemical marker of environmental pollution and exposure to PAC. Carp hepatic 7-pentoxyresorufin O-depentylase (PROD) was unsuitable as a specific indicator of elevation of cytochrome P4502B-like isoenzymes (and thus as a biomarker of presence of CYP2B-type of inducers e.g. organochlorine pesticides) due to a probable inducibility by CYP1A inducers. PROD, unlike aminopyrine N-demethylase, another mammalian CYP2B-dependent activity which was not increased by exposure to PAC, was strongly inhibited by CYP1A inhibitors, α-naphthoflavone and 9-hydroxyellipticine. The respective dose-response curves were related to the EROD inhibition. Chloramphenicol, an inhibitor of CYP2B enzymes. inhibited both activities only weakly or not at all. Adaptation of carp to lower ambient temperatures and to the stress associated with reduction of water volume and subsequent capture by netting proved to highly significantly inhibit levels of EROD and PROD activities. Summer appeared to be the most suitable season for the biochemical monitoring of contamination. Activity of 7-ethoxycoumarin O-deethylase (ECOD) was also markedly induced by PAC, but the enzyme was less responsive than the EROD activity. The ECOD activity was not significantly influenced by ambient temperature or by stress associated with capture and removal of fish. This probably reflected a modulation of partly different CYP enzymes other than EROD. Unlike in ECOD, the results confirmed the high specificity and sensitivity of induction of CYP1A-dependent EROD activity in carp (PAC in environmental exposure levels affected EROD highly significantly).

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, indicatingthe inductionof cytochrome P450 subfamiliesCYP1A 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 xenobioticmetabolizing enzyme system at the site of a primary portal of xenobiotic entry to the systemic circulation.

Alterations of Cytochrome P450-Dependent Monooxygenase Activities in Eriocheir japonicus in Response to Water Pollution

Eriocheir japonicus, fresh-water crabs inhabiting rivers and estuaries in Japan, were investigated for cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activities to see if these activities reflect the river pollution gradient. From the laboratory dose-response experiments, we found that the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene induced total CYP contents, ethoxycoumarin O-deethylase activity, and bunitrolol 4-hydroxylase activity in crab hepatopancreas. In the field studies, crabs collected from the river with the highest concentration of PAHs exhibited the highest levels of CYP, the highest activities of benzo[a]pyrene 3-hydroxylase, imipramine 2-hydroxylase, bunitrolol 4-hydroxylase, ethoxycoumarin O-deethylase, and the ability to metabolically activate benzo[a]pyrene, but erythromycin N-demethylase activity was not induced. The correlation between PAH levels and drug-metabolizing enzyme activities in female crabs were not as marked as in male crabs. The levels and activities of CYP did not appear to reflect the concentrations of organochlorines and polychlorinated biphenyl congeners (PCBs) studied in the fat of crab hepatopancreas.

Alterations of cytochrome P450-dependent monooxygenase activities in Eriocheir japonicus in response to water pollution.

Eriocheir japonicus, fresh-water crabs inhabiting rivers and estuaries in Japan, were investigated for cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activities to see if these activities reflect the river pollution gradient. From the laboratory dose-response experiments, we found that the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene induced total CYP contents, ethoxycoumarin O-deethylase activity, and bunitrolol 4-hydroxylase activity in crab hepatopancreas. In the field studies, crabs collected from the river with the highest concentration of PAHs exhibited the highest levels of CYP, the highest activities of benzo[a]pyrene 3-hydroxylase, imipramine 2-hydroxylase, bunitrolol 4-hydroxylase, ethoxycoumarin O-deethylase, and the ability to metabolically activate benzo[a]pyrene, but erythromycin N-demethylase activity was not induced. The correlation between PAH levels and drug-metabolizing enzyme activities in female crabs were not as marked as in male crabs. The levels and activities of CYP did not appear to reflect the concentrations of organochlorines and polychlorinated biphenyl congeners (PCBs) studied in the fat of crab hepatopancreas.

Activities of liver and lung cytochrome P450-dependent monooxygenases and antioxidant enzymes in laboratory and wild Norway rats exposed to reference and contaminated soils.

Laboratory and wild Norway rats were exposed in the laboratory to an uncontaminated soil and to a soil from a site contaminated with petrochemical waste. Activities of microsomal lung and liver cytochrome P450-dependent monooxygenases, including 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD) and 7-benzoxyresorufin O-debenzylase (BROD) were measured at selected times during the course of the study. The highest degree of induction of hepatic EROD (7-fold) was shown after 3 days of exposure to the contaminated soil. However, two months later, the EROD activity declined to fourfold increase over the control. The PROD and BROD activities displayed a similar time course of induction, but the degree of induction was lower. The induction of hepatic monooxygenase activities was observed in both laboratory and wild rats. Lung monooxygenase EROD was highly induced (up to 28-fold) after 3 days of exposure, and the activity remained elevated throughout the two-month experiment. BROD and PROD activities were not induced. The activities of three antioxidant enzymes, namely superoxide dismutase, glutathione peroxidase (Se- and non-Se- dependent) and catalase also were measured in lung and liver cytosol, but no significant changes were observed after two months of exposure to contaminated soil.

Activities of Liver and Lung Cytochrome P450-Dependent Monooxygenases and Antioxidant Enzymes in Laboratory and Wild Norway Rats Exposed to Reference and Contaminated Soils

Activities of Liver and Lung Cytochrome P450-Dependent Monooxygenases and Antioxidant Enzymes in Laboratory and Wild Norway Rats Exposed to Reference and Contaminated Soils

Activities of cytochrome P450-dependent monooxygenases and antioxidant enzymes in different organs of Norway rats ( Rattus norvegicus) inhabiting reference and contaminated sites

Wild Norway rats ( Rattus norvegicus) were collected from a site contaminated by a range of polycyclic aromatic hydrocarbons (PAHs), mineral oils, polychlorobiphenyls (PCBs) and heavy metals. Activities of cytochrome P450 monooxygenases (ethoxy-, pentoxy- and benzoxy-resorufin O-dealkylases, and 4-nitrophenol hydroxylase) were measured in microsomal fractions from liver and lung. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and non-selenium-dependent glutathione peroxidases) were also measured in cytosolic fractions from lung and liver, and in erythrocytes. The levels of activities were compared with those found in control laboratory rats and in wild Norway rats reared in a terrarium. Results show that rats living in a polluted environment have monooxygenase activities higher than that of control animals in both liver and lung. Some modifications of antioxidant enzyme activities were also found in these animals.

Difference between in vivo and in vitro effects of propofol on defluorination and metabolic activities of hamster hepatic cytochrome P450-dependent mono-oxygenases

We have compared the in vivo and in vitro effects of propofol on cytochrome P450-dependent monooxygenase activities in hamster liver microsomes. Propofol (Diprivan) 10 mg/100 g body weight was injected i.p. twice a day for 2 weeks to induce cytochrome P450 enzymes. Liver microsomes were prepared by differential centrifugation. Metabolism of the cytochrome P450-dependent mono-oxygenase system was evaluated by measuring aniline hydroxylation, benzphetamine demethylation and benzo(a)pyrene hydroxylation. Defluorination of enflurane was assayed by detecting free fluoride metabolites. At similar concentrations as in the in vivo group, propofol in vitro exhibited concentration-dependent inhibition of metabolism of benzphetamine and benzo(a)pyrene. Aniline hydroxylation and defluorination of enflurane were inhibited to 78% of control with propofol 0.25 mmol litre-1. In propofol-treated hamsters, there was only minimal inhibitory or inductive effects on either mono-oxygenase activities or capacity for defluorination. This difference between the in vitro and in vivo effects of propofol on cytochrome P450 mono-oxygenase activities emphasizes the need for care when comparing in vitro and clinical data.

3,3′,4,4′‐Tetrachlorobiphenyl Effects on Antioxidant Enzymes and Glutathione Status in Different Tissues of Rainbow Trout

Polychlorinated biphenyls are known to cause induction in cytochrome P450-dependent monooxygenase activities and alteration in the antioxidant defense of mammals. To determine whether similar detoxication processes are activated in rainbow trout (Oncorhynchus mykiss), we investigated P450-dependent enzyme activities, antioxidant enzymes and glutathione status (reduced and oxidized glutathione, GSH and GSSG) in this species injected intraperitoneally with 3,3',4,4'-tetrachlorobiphenyl at 5 mg/kg body weight 6 weeks post injection. Ethoxyresorufin O-deethylase activities increased 11- and 40-fold in liver and kidney. UDPglucuronosyltransferase activities were 2- and 5-fold higher in these organs, while glutathione S-transferase activity was enhanced greater than 2-fold in liver of tetrachlorobiphenyl injected trout in comparison with controls. Glutathione peroxidase activities were increased in liver and white muscle of dosed fish. Tetrachlorobiphenyl exposure resulted in a significant increase in glutathione reductase activities, with 7-fold enhancement in liver and significantly elevated activities in kidney, red and white muscles. Similarly, cytosolic superoxide dismutase and catalase activities were increased in white muscle of injected trout. Tetrachlorobiphenyl exposure significantly increased GSH concentrations in liver and kidney, while GSSG levels were increased in liver and blood plasma. These changes, however, did not modify the GSSG/GSH ratios in these tissues. Overall, these results imply a major tetrachlorobiphenyl effect on GSH status and antioxidant enzymes in trout tissues and identify white muscle along with liver and kidney as important tissues in the detoxication process in this animal.

Selective increase of rat lung cytochrome P450 1A1 dependent monooxygenase activity after acute sodium arsenite administration.

Arsenic is a known pulmonary, hepatic, and skin carcinogen in humans and a known inducer of stress proteins. Consequently, the ability of arsenite (As3+) to modulate isozyme-selective cytochrome P450 (P450) dependent monooxygenase activities was investigated in microsomes prepared from lung, liver, and kidney of male, adult Sprague-Dawley rats treated subcutaneously (s.c.) with sodium arsenite (75 mumol/kg body weight) 24 h before death. In the lung, the activity of P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation (ERFD) was markedly (approximately 5-fold) increased in treated versus control rats, whereas the activity of P450 2B catalyzed 7-pentoxyresorufin O-depentylation (PRFD) was unchanged. Pulmonary ERF activity remained elevated for at least 48 h after As3+ treatment. In contrast, As3+ inhibited hepatic microsomal ERFD and PRFD activity by approximately 20 and 35%, respectively, 24 h after treatment. ERFD activity was also decreased in kidney microsomes of As(3+)-treated rats, but the inhibition was greater than in liver (50 vs. 35%) 24 h after injection. These effects are almost certainly not due to a direct action of As3+ on P450-dependent catalysis, as in vitro addition of sodium As3+ at concentrations up to 1 mM had no effect on ERFD activity of control rat lung microsomes. In addition, pretreatment of rats with Zn (153 mumol.kg-1.day-1 for 2 days, s.c.) had no effect on control or As(3+)-mediated changes in P450-dependent ERFD activity of rat lung or kidney microsomes. These results demonstrate that As3+ is an isozyme-selective modulator of P450 monooxygenase activity (i.e., significant increase of P450 1A1 catalyzed activity but not P450 2B catalyzed activity) in rat lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of hepatocarcinogenesis and induction of specific cytochrome P450-dependent monooxygenase activities by the barbiturates allobarbital, aprobarbital, pentobarbital, secobarbital and 5-phenyl- and 5-ethylbarbituric acids.

To test predictions that barbiturates which are long-acting sedatives and/or strong inducers of CYP2B-mediated monooxygenase activities would be effective promoters of hepatocarcinogenesis, a series of clinically-useful barbiturates and structural analogs were tested for ability to promote hepatocellular carcinogenesis in male F344/NCr rats initiated with N-nitrosodiethylamine and for efficacy as inducers of CYP2B activity in non-initiated rats of the same sex and strain. The barbiturates were administered in the diet at concentrations equimolar to 500 p.p.m. of the known liver tumor promoter phenobarbital, which served as the positive control for this study. Phenobarbital, which has the longest duration of sedative action of this series of compounds, caused the greatest induction of CYP2B activity, and displayed strong liver tumor promoting effects. Allobarbital and aprobarbital, two intermediate-duration sedatives, were found to promote hepatocarcinogenesis, with allobarbital proving to be as effective as phenobarbital in this respect and aprobarbital being somewhat weaker as a promoter. These intermediate-duration sedatives were each relatively weak CYP2B-type inducers, causing approximately 25% of the induction displayed by phenobarbital. The nonsedatives, 5-phenyl- and 5-ethyl-barbituric acids, were essentially inactive as CYP2B-type inducers and were also found to be relatively inactive as promoters of hepatocarcinogenesis. Of the shorter-duration sedatives, pentobarbital was found to promote, and was relatively effective as a CYP2B-type inducer, while secobarbital showed little or no promoting activity and was less effective as an inducer of CYP2B activities. Pentobarbital thus proved an important exception to our hypothesis that only long-acting sedative barbiturates would promote hepatocarcinogenesis. Although both the durations of sedative action and the degrees of CYP2B-type induction exhibited by these compounds correlate with a quantitative parameter for liver tumor-promoting activity (relative promotion index), neither parameter appears to be sufficient, by itself, as a predictor of promoting activity for rat liver.

Induction of CYP2B1 and 3A1, and associated monooxygenase activities by tamoxifen and certain analogues in the livers of female rats and mice

Previous studies suggest long-term feeding of tamoxifen { Z-1-[4-(2-dimethylaminoethoxy)phenyl]1, 2-diphenyl-1-butane} to rats gives rise to liver tumours, while mice are resistant. The effects of tamoxifen on cytochrome P450 isoenzymes and associated monooxygenase activities in the livers of female Fischer rats and C57B1/6 and DBA/2 mice have been compared. Total microsomal cytochrome P450 was not induced in the livers of rats given tamoxifen (45 mg/kg daily for 4 days) and was in fact significantly reduced after 3 days treatment. In contrast, there was a 30–60-fold increase in the metabolism of benzyloxy- and pentoxyresorufins to resorufin. Ethoxyresorufin O-deethylase was induced only 2.5-fold. The regio- and stereo-specific hydroxylation of testosterone following tamoxifen pretreatment of rats showed a general time- and dose-dependent induction. 6β- and 16α-hydroxylation of testosterone together with oxidation to androstenedione were increased 2–3-fold while 2β-hydroxylation was induced only marginally, suggesting that tamoxifen produces a mixed pattern of induction with a significant phenobarbitone-like component. No induction of the 2β- or 6β-hydroxylation pathway occurred in either mouse strain. In rats, immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or 3A1 showed that tamoxifen pretreatment resulted in 2–3-fold increases in both CYP2B1, 2B2 and 3A1 proteins, relative to controls. Immunohistochemistry of rat liver sections showed a centrilobular localization of these induced proteins. Similar patterns of induction as measured by immunoblotting experiments and testosterone hydroxylation were seen following the administration of structurally related analogues, toremifene and droloxifene (3-hydroxytamoxifen), thought to be noncarcinogenic in the rat. No induction of these monooxygenase activities was seen in C57B1/6 mice and only small increases in benzyloxy- and pentoxyresorunn metabolism were in DBA/2 mice. It is suggested that the induction of cytochrome P450-dependent activities by tamoxifen may result in accelerated liver metabolism of this drug with important implications for the disposition of tamoxifen in vivo and also for its metabolic conversion to genotoxic metabolite(s). The difference in inducibility of cytochrome P450-dependent monooxygenase activities between rats and mice offers a plausible and testable hypothesis that the difference in tamoxifen metabolism between the two species may contribute to their carcinogenic response to tamoxifen.

Regulation of Cytochrome P450 in Cultured Human Colonic Cells

The consequences of altered cytochrome P450-dependent monooxygenase activities in colonic tissue are unknown. As an initial step toward elucidating underlying mechanisms that regulate cytochrome P450 levels in colonic epithelium, we have characterized CYP1A13 induction in cultured human colonic cells (Caco-2). The induction of CYP1A1 by several inducers was measured by enzymatic activity and immunoreactivity. 3-Methylcholanthrene, β-naphthoflavone, and benz[a]anthracene were strong inducers of CYP1A1, benzo[a]pyrene induced CYPlAl activity only weakly, while benzo[a]pyrene and phenobarbital were essentially inactive as inducers. The response of Caco-2 cells to β-naphthoflavone is similar to that previously observed in vivo in the rat colonic epithelium. In addition, we have demonstrated that this induction results from an increase in CYP1A1 transcriptional activity. These results suggest that the Caco-2 cell line exhibits certain characteristics of cytochrome P450 activity observed in colonic epithelium and may serve as a model for studying cytochrome P450-dependent activity in this tissue. The identification of a colonic cell line that responds to Xenobiotic inducers will be useful for examining mechanisms of cytochrome P450 induction in the colon and elucidating the potential role of this system in colonic carcinogenesis.