Identification of N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine as the major adduct formed by the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, with DNA.

The covalent binding of the N-acetoxy-, N-hydroxy-, and nitro derivatives of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to 2'-deoxyribonucleosides or DNA was investigated in vitro and in vivo. N-Acetoxy-PhIP reacted with deoxyguanosine (dG), but not with the other deoxyribonucleosides, to form N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP), whose structure was determined by NMR and mass spectral analyses and by ultraviolet absorption and pH-solvent partitioning characteristics. While reaction of N-acetoxy-PhIP with calf thymus DNA at pH 5.0 yielded 5.38 +/- 1.16 nmol of bound PhIP residues/mg of DNA, N-hydroxy-PhIP gave only 0.13-0.23 nmol binding/mg of DNA under identical reaction conditions. Nitro-PhIP produced no detectable binding under these conditions. HPLC analysis of 1-butanol extracts of enzymatically hydrolyzed DNA that had been modified by N-acetoxy-PhIP in vitro showed a major adduct which coeluted with and had an ultraviolet absorption and a mass spectrum that were identical to that of authentic dG-C8-PhIP. 32P-Postlabeling analysis of DNA isolated from colon, pancreas, lung, heart, and liver of rats treated orally with PhIP revealed the presence of a major PhIP-DNA adduct. This adduct had chromatographic properties identical to that of the 32P-labeled bis(phosphate) derivative of dG-C8-PhIP and represented 35-45% of the total adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel LC-ESI/MS/MS(n) method for the characterization and quantification of 2'-deoxyguanosine adducts of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by 2-D linear quadrupole ion trap mass spectrometry

An accurate and sensitive liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS(n)) technique has been developed for the characterization and quantification of 2'-deoxyguanosine (dG) adducts of the dietary mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). PhIP is an animal and potential human carcinogen that occurs in grilled meats. Following enzymatic digestion and adduct enrichment by solid-phase extraction (SPE), PhIP-DNA adducts were analyzed by MS/MS and MS(n) scan modes on a 2-D linear quadrupole ion trap mass spectrometer (QIT/MS). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP), was detected in calf thymus (CT) DNA modified in vitro with a bioactivated form of PhIP and in the colon and liver of rats given PhIP as part of the diet. The lower limit of detection (LOD) was 1 adduct per 10(8) DNA bases, and the limit of quantification (LOQ) was 3 adducts per 10(8) DNA bases in both MS/MS and MS(3) scan modes, using 27 microg of DNA for analysis. Measurements were based on isotope dilution with the internal standard, N-(deoxyguanosin-8-yl)-2-amino-1-(trideutero)methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-[2H3C]-PhIP). The selected reaction monitoring (SRM) scan mode in MS/MS was employed to monitor the loss of deoxyribose (dR) from the protonated molecules of the adducts ([M + H - 116]+). The consecutive reaction monitoring (CRM) scan modes in MS(3) and MS(4) were used to measure and further characterize product ions of the aglycone ion (BH2+) (Guanyl-PhIP). The MS(3) scan mode was effective in eliminating isobaric interferences observed in the MS/MS scan mode and resulted in an improved signal-to-noise (S/N) ratio. Moreover, the product ion spectra obtained by the MS(n) scan modes provided rich structural information about the adduct and were used to corroborate the identity of dG-C8-PhIP. In addition, an isomeric dG-PhIP adduct was detected in vivo. This LC-ESI/MS/MS(n) method is the first reported application on the use of the MS(3) scan mode for the analysis of DNA adducts in vivo.     

Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice.

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20-30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P < 0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6+/-0.6 vs 4.6+/-0.7, respectively, relative adduct labeling (RAL) x 10(8), mean+/-S.E.M., n = 3-5 mice). In the mammary gland, however, there was no difference in IQ-DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP-DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP-DNA adducts in liver and kidney of WT mice were 9.9+/-1.1 and 22.5+/-6.9, respectively, whereas no PhIP-DNA adducts were detected in either organ of KO mice (limit of detection, 1.4-2.8 x 10(9)). PhIP-DNA adduct levels in mammary gland and colon of WT mice were 47.1+/-9.5 and 58.0+/-21.7, respectively, but accordingly only 3.8+/-0.7 and 5.4+/-0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.     

Chemical synthesis of 2'-deoxyguanosine-C8 adducts with heterocyclic amines: an application to synthesis of oligonucleotides site-specifically adducted with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Synthesis of 2'-deoxyguanosine-C8 adducts (dG-C8 adducts) with mutagenic/carcinogenic heterocyclic amines (HCAs) was achieved via the Buchwald-Hartwig arylamination reaction. By using tris(dibenzylideneacetone)dipalladium (Pd(2)dba(3)) and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (xantphos) with a cesium carbonate (Cs(2)CO(3)) base at a reaction temperature of 100 approximately 120 degrees C, we obtained derivatives of dG-C8 adducts with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in 69% approximately 97% yield from the cross-coupling of an 8-bromodeoxyguanosine derivative. In the case of PhIP, it was found that dimethyl sulfoxide (DMSO) was the critical solvent for the arylamination reaction. Subsequent deprotection of the resulting dG-C8 adduct derivatives yielded authentic samples of dG-C8 adducts with HCAs. The dG-C8-PhIP adduct was further converted into a suitably protected phosphoramidite derivative for automated DNA synthesis. Synthesis of oligonucleotides wherein PhIP adducted on each G within a triple G sequence in codon 869 (TCC GGG AAC) of rat Apc genes was performed with a modification in the coupling time and deprotection procedures.     

Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats.

The chemopreventive properties of dietary indole-3-carbinol (I3C) were evaluated by assessing its effect on DNA adduct formation and metabolism of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and the induction of cytochromes P450 1A1 and -1A2 in female F344 rats. In experiment 1, animals on I3C diets (0, 0.02% or 0.1%, w/w) were treated by gavage with 1mg/kg/day of PhIP for 23 days. On days 2, 9, 16 and 23, their 24-hr urine was collected and unmetabolized PhIP was measured by GC/MS. On day 24, the animals were sacrificed, and DNA from pancreas, spleen, white blood cells (WBCs), lung, colon, kidney, mammary epithelial cells, caecum, heart, small intestine, liver and stomach was isolated for determination of PhIP-DNA adduct levels by (32)P-postlabelling assays. Except in the mammary gland, I3C diets significantly inhibited PhIP-DNA adduct formation in WBCs and in all organs, ranging from 34.7 to 67.7% with the 0.02% I3C diet to 68.4 to 95.3% with the 0.1% I3C diet. I3C diets also significantly decreased the concentration of urinary unmetabolized PhIP to 29.5-38.4% (0.02% I3C) and 12.8-17.8% (0.1% I3C) of values obtained with the I3C-free diet. In experiment 2, animals were either treated by intubation of I3C at 100 or 200mg/kg for 2 consecutive days or given an I3C-containing diet (0.02% or 0.1%, w/w) for 2 weeks. The expression and activity of cytochromes P450 1A1 and -1A2 were studied by Northern blots, Western blots, and in vitro enzyme determinations. Both the expression and activity of these cytochromes were induced by all of the I3C treatments. It is concluded that, in the female F344 rat, dietary I3C inhibits PhIP-DNA adduct formation and accelerates PhIP metabolism, probably through induction of cytochromes P450 1A1 and -1A2. The chemopreventive properties of I3C in PhIP-induced carcinogenesis are probably mediated through enhancement of PhIP detoxification pathways.     

Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: studies with the hepatic NADPH:cytochrome P450 reductase null mouse

Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by (32)P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.     

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and (32)P-postlabelling

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kgbody weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.     

In vivo induction of human cytochrome P450 enzymes expressed in chimeric mice with humanized liver.

The induction and inhibition of human cytochrome P450 (P450) enzymes are clinically responsible for drug interactions. Although the induction of P450s is investigated using human hepatocytes in the drug development process, there are some disadvantages, such as the decline of the enzyme activity during culture. In the present study, we examined the in vivo induction potency in chimeric mice with humanized liver, which was recently established in Japan to clarify whether this chimeric mouse model would be more suitable for human induction studies. Rifampicin and 3-methylcholanthrene (3-MC) were used in vivo as typical P450 inducers in the chimeric mice. The expression levels of human CYP3A4 mRNA and CYP3A4 protein and dexamethasone 6-hydroxylase activity, specific for human CYP3A4, were increased 8- to 22-, 3- to 10-, and 5- to 12-fold, respectively, by treatment with rifampicin. In addition, the expression levels of human CYP1A2 mRNA and CYP1A2 protein were also increased 2- to 9- and 5-fold, respectively, by treatment with 3-MC. Although other human P450s are expressed in the chimeric mice, there were few effects by the treatment of rifampicin and 3-MC on the mRNA, protein, and enzyme activity of those P450s. It was demonstrated that human P450s expressed in the chimeric mice with humanized liver were induced by rifampicin and 3-MC. This chimeric mouse model may be a useful animal model to estimate and predict the in vivo induction of P450s in humans.     

DNA adduct formation of 4-aminobiphenyl and heterocyclic aromatic amines in human hepatocytes

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation.     

Dietary omega-3 fatty acids as potential inhibitors of carcinogenesis: effect on DNA adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice and rats.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is carcinogenic in the CDF1 mouse, causing lymphomas (spleen and lymph nodes) and in the F344 rat, causing mammary tumours in the female and colon tumours in the male. Dietary fish oil, a rich source of omega-3 fatty acids, exhibits chemopreventive properties in several rodent tumour models. The potential chemopreventive properties of dietary omega-3 fatty acid ethyl ester concentrate (O3C) were tested by evaluating its effects on the formation and removal of PhIP-DNA adducts. In the first experiment, a powdered AIN-76A diet containing 4.0% (w/w) O3C inhibited PhIP-DNA adduct formation in various organs of the CDF1 mouse, but not in those of the F344 rat. In a subsequent, second experiment, groups of male CDF1 mice were maintained for 43 days on AIN-76A diets containing the following percentages (w/w) of corn oil ethyl esters and O3C: 7.0 and 0, 5.5 and 1.5, 4.0 and 3.0, and 1.0 and 6.0, respectively. All animals received 0.04% (w/w) PhIP in the diet during weeks 3 and 4. Using 32P-postlabelling assays, PhIP-DNA adducts were analysed in various organs and white blood cells (WBC) on days 1, 8 and 15 after removal of PhIP from the diet. In the liver, O3C-containing diets inhibited adduct formation at all three time points (40.3-60.0%, 53.4-75.7% and 43.3-64.3% on days 1, 8 and 15, respectively). In the spleen, inhibition was evident only on days 8 (35.4-38.8%) and 15 (38.4-56.5%). O3C diets inhibited adduct formation in the stomach, small intestine and caecum at all three time points (except in the stomach and caecum on day 15) amounting to 18.5-31.5% decreases in the stomach, 40.0-60.3% decreases in the small intestine and 24.4-31.4% decreases in the caecum. The extent of inhibition was not related to O3C concentration. In the colon and WBC, adduct levels were independent of the type of diet. In all organs, adduct levels decreased significantly over time, with day 15 levels being 6.3-31.6% of those on day 1. Rate of adduct removal was independent of the type of diet. It is concluded that dietary O3C inhibits PhIP-DNA adduct formation in a target organ (spleen) as well as in non-target organs (liver and gastrointestinal tract) of the CDF1 mouse, but that the rate of adduct removal is independent of the O3C content of the diet.     

Mass spectrometric characterization of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine N-oxidized metabolites bound at Cys34 of human serum albumin

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed during the cooking of meats and poultry. PhIP is a carcinogen in rodents and a potential human carcinogen. Several short-term biomarkers of PhIP have been established for human biomonitoring, but validated long-term biomarkers of the biologically effective dose of PhIP remain to be developed. Metabolites of PhIP have been reported to covalently bind to human serum albumin (SA), which is the most abundant protein in plasma; however, the chemical structures of PhIP-SA adducts are unknown. Cysteine(34) is one of 35 conserved Cys residues in SA across species. Thirty-four of these Cys are involved in 17 disulfide bonds. The single unpaired Cys(34) residue in SA is well-known to react with carcinogenic metabolites and toxic electrophiles. 2-Nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO(2)-PhIP), 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), and 2-nitroso-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO-PhIP), three genotoxic metabolites of PhIP, were reacted with purified human SA or human plasma, and the SA adduction products, following enzymatic digestion, were separated by ultra performance liquid chromatography and characterized with a linear quadrupole ion trap mass spectrometer. The major adduct of NO(2)-PhIP was formed at the Cys(34) of SA with bond formation occurring between the sulfhydryl group of Cys and the C-2 imidazole atom of PhIP. The major adducts formed between SA and HNOH-PhIP or NO-PhIP were identified as acid-labile sulfinamide linkages at Cys(34). These PhIP-SA adducts represent a measure of bioactivation of PhIP and may serve as long-term biomarkers of the biologically effective dose of PhIP.     

Small intestinal cytochromes P450.

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally--being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics--with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid--aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly--iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.     

Small Intestinal Cytochromes P450

Abstract

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally — being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics — with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid — aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly — iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.     

The inducibility and catalytic activity of cytochromes P450c (P450IA1) and P450d (P450IA2) in rat tissues

The metabolism of phenacetin is primarily by cytochrome P450-dependent O-deethylation to paracetamol (POD activity). In untreated rats, microsomal POD activity is detectable in both the liver and lung, but not in the small intestine or the kidney. POD activity is highly induced in both hepatic and extrahepatic tissues of the rat following treatment with polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC). Only cytochrome P450c (P450IA1) is inducible in rat extrahepatic tissues by MC or isosafrole, whereas in the liver both cytochromes P450c and P450d (P450IA2) are inducible by these compounds. Specific antibodies to cytochromes P450c and P450d were used to study the expression and function of these two related isoenzymes in rat liver and extrahepatic tissues before and after induction with MC. Whereas cytochrome P450d is responsible for all of the high affinity POD activity in hepatic microsomal fractions of both untreated and MC treated rats, this activity is mediated only by P450c in microsomal fractions from extrahepatic tissues following MC treatment. POD activity of microsomal fractions from lung of untreated rats was not mediated by either cytochrome P450c or P450d.     

CYP450氧化还原酶的药物基因组学研究进展

细胞色素P450氧化酶(cytochrome P450enzymes,CYP)的氧化还原反应是人体内重要的生理生化反应,参与许多内、外源化合物的代谢和激素类化合物的合成.CYP450氧化还原酶(cytochrome P450 oxidoreductase,POR)是所有肝微粒体内CYP酶的唯一电子供体.POR不仅可作为电子供体参与由CYP介导的药物代谢,而且可通过1-电子还原反应直接介导一些抗肿瘤前体药物的代谢和转化.可见,POR在药物代谢过程中发挥着极其重要的作用.众多研究证实,编码人POR的基因具有遗传多态性,对临床药物代谢乃至疗效有着显著影响,具有重要的临床意义.下面对近年来POR的药物基因组学最新研究进展作一综述.     

The hepatic cytochrome P450 reductase null mouse as a tool to identify a successful candidate entity

Cytochrome P450s (CYP) play a pivotal role in the metabolism of drugs and xenobiotics, and have been intensively studied over many years. Much of the work carried out on the role of hepatic cytochrome P450s in drug metabolism and disposition has been done in vitro, and has yielded vital information on P450 regulation and function. However, additional factors such as route of administration, absorption, drug transporters, renal clearance and extra-hepatic P450s, make it difficult to extrapolate from in vitro data to in vivo pharmacokinetics. A number of cytochrome P450s knockout mice have been generated, although many have been of limited usefulness due to either embryonic/perinatal lethality, or the functional redundancy inevitably found in a large family of isoenzymes. We have developed a mouse line (HRN) in which cytochrome P450 oxidoreductase (POR), the unique electron donor to cytochrome P450s is deleted specifically in the liver, resulting in the loss of essentially all hepatic P450 function. The HRN mouse, although having disturbances in lipid and bile acid homeostasis develops and breeds normally. We have used the HRN mouse as a model to establish the role of hepatic versus extra-hepatic metabolism in drug metabolism and disposition, and also to investigate the relationship between drug toxicokinetics and therapeutic effect, initially with the chemotherapeutic prodrug cyclophosphamide (CPA).     

Role of cytochrome P450 in hepatotoxicity induced by di- and tributyltin compounds in mice

 The role of cytochrome P450 in the induction of hepatotoxicity by butyltin compounds such as tributyltin chloride (TBTC) and dibutyltin dichloride (DBTC) was investigated in vivo. The pretreatment of mice with SKF-525A, which decreased hepatic levels of cytochrome P450, suppressed TBTC-induced hepatotoxicity, as estimated by serum ornithine carbamyl transferase activity, whereas pretreatment with phenobarbital (PB), which increased the levels of cytochrome P450, enhanced the hepatotoxicity of TBTC. In the case of DBTC, PB pretreatment enhanced hepatotoxicity, while SKF-525A had no effect. Under these experimental conditions only PB pretreatment was found to increase hepatic levels of tin in mice treated with TBTC. These results suggest that hepatic metabolism of butyltin compounds by cytochrome P450 is more closely related to the induction of hepatotoxicity by TBTC than by DBTC. The active tin compounds formed during hepatic metabolism, which are responsible for induction of hepatotoxicity, will be discussed Received: 20 December 1994/Accepted: 20 April 1995     

Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen

1. The mutagenicity of 1-nitropyrene metabolites in Chinese hamster ovary (CHO) cells, in the absence of rat liver S9, decreased in the order 6-hydroxy-1-nitropyrene > 1-nitropyrene 9,10-oxide > 1-nitropyrene 4,5-oxide ～ 3-hydroxy-1-nitropyrene ～ 8-hydroxy-1-nitropyrene > 1-nitropyrene. The order of mutagenicity with rat liver S9 was 1-nitropyrene 4,5-oxide ～ 6-hydroxy-1-nitropyrene ～ 1-nitropyrene 9,10-oxide > 3-hydroxy-1-nitropyrene ～ 1-nitropyrene > 8-hydroxy-1-nitropyrene.

2. 1-Nitropyrene 4,5-oxide reacted with calf thymus DNA to give one or several closely related adducts. The same adducts were detected in CHO cells incubated with 1-nitropyrene 4,5-oxide. Inclusion of a nitroreductase, xanthine oxidase, in the incubations with calf thymus DNA resulted in the formation of an additional adduct identified as N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP).

3. 1-Nitropyrene 9,10-oxide reacted with calf thymus DNA to give an adduct pattern similar to that observed with 1-nitropyrene 4,5-oxide. Incubation of 1-nitropyrene 9,10-oxide with CHO cells resulted in the formation of the same adducts along with dG-C8-AP.

4. dG-C8-AP and N-(deoxyguanosin-8-yl)-1-amino-x-nitropyrene (x = 3, 6 or 8; dG-C8-ANP) were detected in injection site DNA from Sprague-Dawley rats treated with 1-nitropyrene. In mammary gland DNA, dG-C8-AP and an unidentified adduct were found. dG-C8-ANP was the only DNA adduct detected in the livers of newborn CD-1 mice and the lungs of A/J mice dosed with 1-nitropyrene.     

The application of hepatic P450 reductase null gpt delta mice in studying the role of hepatic P450 in genotoxic carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mutagenesis

The cytochrome P450 (P450 or CYP) is involved in both detoxification and metabolic activation of many carcinogens. In order to identify the role of hepatic P450 in the mutagenesis of genotoxic carcinogens, we generated a novel hepatic P450 reductase null (HRN) gpt delta mouse model, which lacks functional hepatic P450 on a gpt delta mouse background. In this study, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was used to treat HRN gpt delta mice and control littermates. Gene mutations in the liver and lungs were detected, and mutation spectra were analyzed. Pharmacokinetic analyses were performed, and tissue levels of NNK and metabolite were determined. NNK-induced mutant frequencies (MFs) were equivalent to spontaneous MFs in the liver, but increased more than 3 times in the lungs of HRN gpt delta mice compared to control mice. NNK-induced mutation spectra showed no difference between HRN gpt delta mice and control littermates. Toxicokinetic studies revealed reduced clearance of NNK with elevated tissue concentrations in HRN gpt delta mice. To our knowledge, these are the first data demonstrating that NNK cannot induce mutagenesis in the liver without P450 metabolic activation, but can induce mutagenesis in lungs by a hepatic P450-independent mechanism. Moreover, our data show that hepatic P450 plays a major role in the systemic clearance of NNK, thereby protecting the lungs against NNK-induced mutagenesis. Our model will be useful in establishing the role of hepatic versus extrahepatic P450-mediated mutagenesis, and the relative contributions of P450 compared to other biotransformation enzymes in the genotoxic carcinogens’ activation.