Low-level biological dosimetry of heterocyclic amine carcinogens isolated from cooked food.

The bioavailability and the bioreactivity of the carcinogenic heterocyclic amine [2-14C]2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) have been investigated at a dose approximating that likely from the human diet by accelerator mass spectrometry (AMS). [2-14C]PhIP was administered to mice at a dose equivalent ot the consumption of two 100 g beef patties (41 ng/kg). The biological half-life of PhIP was 1 hr, with 90% of the dose being excreted via the urine. Peak tissue PhIP concentrations were reached within 3 hr, with the highest levels in the tissues of the gastrointestinal tract, followed by the liver, kidney, pancreas, and thymus. Since the detection limit by AMS is dependent on the natural abundance of 14C, we have achieved further increases in sensitivity by producing mice that have 20% of the natural abundance of 14C. Use of these 14C-depleted animals allows measurements to be made near the natural level of exposure for many environmental carcinogens. PhIP-DNA adduct levels have also been measured by 32P-postlabeling at doses of 1.0, 10, and 20 mg/kg. The highest adduct levels were found in the pancreas, thymus, heart, and liver and increased linearly with dose. The principal adducts are derived from guanine.

Role of sulfation and acetylation in the activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine to intermediates which bind DNA.

Mutagenic activity associated with amino-imidazoazaarene food-derived mutagens such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) appears to be dependent upon N-hydroxylation, though additional metabolic pathways may be involved in the production of the ultimate reactive intermediate which covalently binds DNA. We have evaluated the ability of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) to bind DNA in vitro and have determined which secondary metabolic pathways are involved in the production of electrophilic intermediates. Incubation of DNA with 10 microM N-hydroxy-PhIP alone or with mouse-liver cytosol did not result in detectable adduct formation. Addition of 3'-phosphoadenosine 5'-phosphosulfate or acetyl coenzyme A to cytosolic incubations containing N-hydroxy-PhIP resulted in DNA adducts which could be detected by 32P-postlabeling at levels of 594 and 30 fmoles/micrograms DNA, respectively. Addition of 3'-phosphoadenosine 5'-phosphosulfate and to a lesser extent acetyl coenzyme A to cytosolic incubations also increased the rate of degradation of the unstable N-hydroxy-PhIP intermediate. These data suggest that both sulfation- and acetylation-dependent metabolic pathways may be important in the mammalian genotoxic actions of PhIP.

Comparison of 3H- and 125I-radioimmunoassay and gas chromatography/mass spectrometry for the determination of delta 9-tetrahydrocannabinol and cannabinoids in blood and serum.

Blood and serum specimens from 100 subjects in a driving study were tested for the presence of delta 9-tetrahydrocannabinol (THC) by 3H- and 125I-labeled RIA and GC/MS. The specimens were also analyzed for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol by the two RIA methods. Blind quality control specimens containing THC were included with each batch of subject specimens and sent coded to three participating laboratories. The methods were assessed for accuracy, reproducibility, detectability, specificity, and the results correlated. It was found that serum was a better matrix than blood for determination of cannabinoids. The three methods gave parallel but significantly different quantitative results, apparently due to calibration problems. However, each technique was capable of measuring THC concentrations up to three hours after usage.

Activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to mutagenic metabolites.

Metabolism of heterocyclic amines to N-hydroxy intermediates appears critical in the mutagenic and carcinogenic actions of these compounds. We have studied the murine hepatic microsomal and cytosolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine mutagen formed in cooked meats. PhIP (25 microM) was almost completely converted to N-hydroxy-PhIP and 4'-hydroxy-PhIP in 30 min by reaction with 3-methylcholanthrene-induced microsomal preparations. Microsomal formation of the active N-hydroxy-PhIP metabolite was slightly favored over the 4'-hydroxy-PhIP detoxification product at all concentrations studied (25-200 microM). Metabolism of PhIP in microsomal preparations derived from control mice was approximately 10% of the induced preparations. Metabolically activated PhIP and synthetic N-hydroxy-PhIP produced concentration-dependent increases in mutagenic activity in both Salmonella strains TA98 and TA98/1,8-DNP6, indicating that acetylated intermediates were not important in the mutagenicity of N-hydroxy-PhIP in these bacteria. Significant stabilization of the N-hydroxy-PhIP intermediate by both microsomal protein and BSA was observed. Addition of cytosol to microsomal incubations with PhIP (25 microM) resulted in an increase in mutagenic activity which could be attributable to stabilization by glutathione. An additional increase in mutagenicity resulted from addition of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), but not acetyl coenzyme A to microsomal preparations containing the cytosolic fraction. Furthermore, addition of PAPS to cytosolic preparations containing synthetic N-hydroxy-PhIP produced a 17% decrease in levels of the arylhydroxylamine relative to controls over 30 min, suggesting that secondary metabolism of N-hydroxy-PhIP to a sulfate conjugate may be relevant to the mutagenic and carcinogenic actions of PhIP.

Metabolism of the food-derived mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in nonhuman primates.

Metabolism of the food-derived heterocyclic amine mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in cynomolgus monkeys. [3H]PhIP (50 mumol/kg, p.o.) was extensively metabolized, with only 1% of the dose excreted into the urine as parent compound. Four metabolites were isolated by HPLC and identified: PhIP-4'-O-glucuronide, PhIP-4'-sulfate, 4'-hydroxy-PhIP and a glucuronide conjugate of N-hydroxy-PhIP. All four metabolites were detected in urine, bile and plasma of monkeys. 4'-Hydroxy-PhIP and PhIP were found in feces. The major PhIP metabolite in urine, bile and plasma was PhIP-4'-sulfate. In urine this metabolite constituted approximately 64-72% of the radioactivity excreted. The clearance of PhIP and PhIP metabolites from plasma was rapid, with the largest elimination occurring within 8 h. Administration of nine consecutive daily doses of unlabeled PhIP (50 mumol/kg, p.o.) prior to administration of [3H]PhIP (50 mumol/kg, p.o.) did not alter the plasma clearance of radiolabeled PhIP or PhIP metabolites, suggesting that this multiple-dose regimen did not induce or alter PhIP metabolism. PhIP formed DNA adducts in white blood cells, as determined by the 32P-postlabeling method. The levels of PhIP-DNA adducts in blood appeared to peak 3 h after administering a single dose of PhIP (50 mumol/kg, p.o.) and were still detected 1 week after dosing. The presence of the glucuronide conjugate of N-hydroxy-PhIP in urine, bile and plasma, and the presence of PhIP-DNA adducts in white blood cells indicate that PhIP undergoes metabolic activation via N-hydroxylation in cynomolgus monkeys. The results suggest that PhIP is activated in vivo to genotoxic metabolites in nonhuman primates and thus is a potential carcinogen in this species.

Analysis of mercapturic acid conjugates of xenobiotic compounds using negative ionization and tandem mass spectrometry.

Mass spectra of mercapturic acid conjugates of two xenobiotic products of lipid peroxidation (trans-4-hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal) as well as conjugates of 1,3-dichloropropene, styrene oxide, 1,2-naphthalene oxide and alpha-chlorotoluene were obtained using fast atom bombardment or negative chemical ionization. Fragmentation pathways were investigated using linked scan and mass-analyzed ion kinetic energy spectrometric techniques. Characteristics of the spectra obtained using different ionization and sample introduction techniques are compared. Deprotonated molecular ions of mercapturic acids gave simple daughter ion spectra, with the dominant mode of decomposition involving cleavage of C-S bonds giving a characteristic neutral loss of 129 Da. Screening for mercapturates in urine samples was performed using neutral loss scanning and yielded limits of detection in the low nanogram per milliliter range. Quantitative analysis of the S-benzyl mercapturic acid at 1 p.p.b. in urine has been demonstrated using combined gas chromatography/electron capture mass spectrometry with d3-S-benzyl mercapturic acid as internal standard.

Pharmacokinetic profiles in rats after intravenous, oral, or dermal administration of dapsone.

Dapsone is a potent anti-inflammatory and antibacterial agent that has been used extensively in the oral treatment of leprosy and dermatitis herpetiformis. This study compared the pharmacokinetic profile of dapsone in rats given a single oral or i.v. 12 mg/kg dose (n = 8/group) or a single dermal application of 12 or 60 mg/kg (n = 12/group) in an aqueous gel application medium containing 10 or 25% diethylene glycol monoethyl ether (DGME). Blood samples (200 microl) were collected via tail vein from each rat and pooled at intervals up to the 24-h period. A terminal blood sample was collected by cardiac puncture from each animal. Plasma concentrations of dapsone were determined by liquid chromatography atmospheric pressure ionization tandem mass spectroscopy. There was no treatment-related overt toxicity observed in any of the animals. Peak levels were reached 1 h after oral dosing (4890 ng/ml), and 6 to 8 h after dermal application, with Cmax values of 1.62, 5.56, and 12.8 ng/ml, for 12 mg/kg at 10 or 25% DGME, and for 60 mg/kg at 25% DGME, respectively. Bioavailability was calculated at 78% after oral dosing and <1% after dermal application. Apparent elimination half-lives (t(1/2))s were similar after i.v. and oral dosing. Both the calculated area under the plasma concentration versus time curve up to 24 h and Cmax values were 3- to 4-fold higher in the dermal application group administered 12 mg/kg dapsone in 25 versus 10% DGME gel, whereas the calculated area under the plasma concentration versus time curve up to 24 h and Cmax values for the 60 mg/kg group were only 3.3- and 2.3-fold greater than those obtained after application of 12 mg/kg in 25% DGME. These results show that both systemic exposure and peak plasma concentrations of dapsone are minimized by dermal versus oral administration of the compound.

Metabolic activation and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in Chinese hamster ovary cells expressing murine cytochrome P450 IA2.

We have utilized Chinese hamster ovary cell lines which stably express a murine cytochrome P450IA2 (P(3)450) cDNA to characterize more fully the mechanisms of genotoxicity of heterocyclic amines derived from cooked meats. To verify that these cell lines were capable of converting promutagens into active metabolites, we studied the microsomal metabolism and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pridine (PhIP). Microsomal preparations derived from excision repair-deficient Chinese hamster ovary cells expressing the mouse cytochrome P(3)450 cDNA (UV5P3) converted PhIP to the genotoxic N-hydroxy-PhIP metabolite. Cytotoxic activity in UV5P3 was observed at concentrations of PhIP as low as 1 microM. Cytotoxicity of PhIP was an order of magnitude lower in a matched repair-proficient cell line (5P3R2) expressing the P(3)450 cDNA. PhIP produced a concentration-dependent increase in sister chromatid exchange (SCE) in UV5P3. N-Hydroxy-PhIP, at concentrations as low as 0.1 microM, produced an increase in SCE in both UV5P3 and in UV5 cells which lack the P(3)450 cDNA. Incubation of PhIP with UV5P3 cells increased the frequency of micronuclei (MN) in cytokinesis-blocked cells. Chromatid gaps, but not aberrations also were induced by treatment with PhIP. N-Hydroxy-PhIP produced increases in MN and chromatid gaps in both UV5 and UV5P3 cell lines; chromosomal aberrations were induced in UV5P3 cells. These results suggested that UV5P3 cells metabolize sufficient quantities of PhIP to produce cytogenetic damage and further indicated that N-hydroxylation of PhIP was requisite for mammalian genotoxicity.

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice.

The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), a heterocyclic amine carcinogen detected in cooked meats, was investigated in mice. In 3-methylcholanthrene-induced mice administered 0.1, 1.0 and 10 mg/kg [14C]PhIP (i.p.), urinary and fecal excretion over 24 h accounted for 16% and 42-56% of the dose respectively. Urinary excretion of unchanged parent compound accounted for only 0.5-0.8% of the administered dose. At all doses, the major urinary metabolite was identified as 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate and this metabolite comprised approximately 5% of the dose. Uninduced mice excreted greater than 13% of a 10 mg/kg dose as the sulfate conjugate. Urinary excretion of both 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) and a glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (N-hydroxy-PhIP) was also higher (4-fold) in uninduced versus induced mice. The decreased urinary excretion of P450-derived metabolites via induction contrasted with increased metabolite formation by hepatic microsomal preparations. 4'-Hydroxy-PhIP and N-hydroxy-PhIP were produced in amounts nearly 7- and 3-fold higher respectively by induced versus uninduced microsomal incubations at 50 microM [3H]PhIP. At concentrations less than 10 microM, PhIP was almost exclusively converted by the induced preparations to an unidentified metabolite that was not retained by the C18 column. This metabolite, which also was formed in incubations with either 4'-hydroxy-PhIP or N-hydroxy-PhIP, was produced by microsomes from uninduced animals at a much slower rate. Covalent binding to microsomal protein in incubations with [3H]PhIP was concentration-dependent and 2- to 4-fold higher in induced than uninduced preparations. Covalent binding in liver and kidney of induced mice administered [14C]PhIP was dose dependent. At 10 mg/kg PhIP, adducts were produced at 1.7-fold higher levels in livers of induced versus uninduced mice, but renal binding was higher in uninduced animals. These studies indicate the importance of cytochrome P450 and other xenobiotic enzymes in the metabolism, disposition and activation of PhIP.

Metabolic processing and disposition of 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in nonhuman primates.

The metabolic processing and disposition of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), three heterocyclic amine mutagens and carcinogens derived from cooked food, was examined in cynomolgus monkeys. IQ is an established hepatocarcinogen in cynomolgus monkeys; however, the carcinogenicity of MeIQx and PhIP is not yet known. IQ was extensively metabolized with little parent compound excreted in urine. Urinary metabolites of IQ arise from a combination of cytochrome P-450 mediated N-demethylation, N-hydroxylation, or ring oxidation at the C-5 position and conjugation with sulfate or glucuronide. IQ was also conjugated at the exocyclic amino group forming IQ-sulfamate and IQ-N-glucuronide. Enteric bacteria biotransformed IQ and its N-demethylated metabolite to 7-oxo-IQ and N-demethyl-7-oxo-IQ, respectively. N-Hydroxy-IQ-N-glucuronide was found in urine of monkeys indicating that metabolic activation via cytochrome P-450-mediated N-hydroxylation occurs in vivo and supporting the theory N-hydroxy-IQ plays a role in the initiation of IQ-induced hepatocarcinogenesis. At least eight urinary metabolites of MeIQx were seen in monkeys fed MeIQx. As was found with IQ, metabolites of MeIQx arise from conjugation with sulfate and glucuronide at the exocyclic amino group, and by cytochrome P-450 mediated oxidation at the C-5 position followed by conjugation with sulfate or glucuronide In contrast to IQ, a significant amount of the dose of MeIQx was excreted unchanged in the urine of monkeys. PhIP was extensively metabolized with a predominant route of metabolism being cytochrome P-450-mediated 4'hydroxylation followed by sulfate conjugation to form PhIP-4'-sulfate. No sulfate conjugation at the exocyclic amino group of PhIP was observed. An N-hydroxy-PhIP-N-glucuronide was also found in urine and bile indicating that metabolic activation of PhIP via N-hydroxylation occurs in vivo in monkeys and suggesting that PhIP may ultimately be carcinogenic to monkeys.

The role of sulfation and/or acetylation in the metabolism of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Salmonella typhimurium and isolated rat hepatocytes.

Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor alpha-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 microM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate), 2-amino-1-methyl-4'-hydroxy-6- phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4'-hydroxy-PhIP to 4'-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) by liver microsomes and isolated rabbit cytochrome P450 isozymes.

The cytochrome P450-dependent metabolism of the heterocyclic amine mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been determined. We investigated the in vitro metabolism of PhIP by polycyclic hydrocarbon-induced mouse and rabbit liver microsomes, and by purified rabbit liver P450 isozymes. Following a 60 min incubation, 3-methylcholanthrene-induced mouse microsomes converted 36% of the PhIP to two major metabolites, N-hydroxy-PhIP and 4'-hydroxy-PhIP, with 43% total metabolism. Rabbit P450 form 6 and form 4 produced the same two major metabolites (20 and 5% total metabolism respectively). Additional metabolites were produced in low yields and amounts varied depending on the isozyme used (1-5%). Metabolites were not detected in incubations of PhIP with P450 forms 2 and 3C. N-Hydroxy-PhIP was found to be directly mutagenic to Salmonella TA98, while the 4'-hydroxy-PhIP was not mutagenic either with or without additional metabolic activation. These data suggest that the cytochrome P450IA isozymes are involved in the metabolism of PhIP by rabbit liver and that formation of N-hydroxy-PhIP is involved in the mutagenicity of PhIP.