Childhood exposure to sexual abuse and partnership outcomes at age 30.

BACKGROUND: In this study, 30-year longitudinal data from the Christchurch Health and Development Study (CHDS) were used to examine the associations between childhood exposure to sexual abuse and intimate relationship outcomes at age 30. In addition, a broad range of early childhood and family confounding factors were tested, and the role of intervening factors from adolescence was explored. METHOD: The investigation analyzed data from a birth cohort of over 900 New Zealand adults studied to the age of 30. At ages 18 and 21 cohort members reported on any exposure to sexual abuse prior to age 16. This information, along with prospective data gathered in childhood and adolescence, was used to predict partnership outcomes at age 30. RESULTS: After adjustment for early childhood and family factors, exposure to more severe forms of childhood sexual abuse (CSA) was associated with earlier and more frequent cohabitation, higher rates of perpetrated interpartner violence (IPV), and early parenthood, lower relationship satisfaction and investment. Several factors from adolescence partially or fully mediated these associations, notably a history of early consensual sexual intercourse, higher number of sexual partnerships, substance abuse problems, and self-esteem. After adjustment for intervening factors, exposure to CSA remained significantly associated with IPV. CONCLUSIONS: The findings support a causal chain process, whereby early childhood and family factors place some individuals at risk for CSA. The extent of CSA exposure is related to adolescent risk taking, which in turn leads to early and more frequent cohabitation, risk of IPV, and lower relationship satisfaction and investment.

Concentration of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) in urine and alkali-hydrolyzed urine after consumption of charbroiled beef.

Heterocyclic amines (HAs) and polycyclic aromatic hydrocarbons are carcinogenic products formed during the cooking of meat at moderate to high temperatures. We have previously shown that the urinary concentration of 1-hydroxypyrene-glucuronide, a metabolite of pyrene, increased significantly in ten subjects who had ingested charbroiled ground beef. We now report the time course and interindividual variation of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) concentration in the urine samples from these ten subjects. PhIP concentration was determined in both untreated and alkali-hydrolyzed urine to obtain estimates of the proportion of conjugated PhIP metabolites in each subject. PhIP was measured by gas chromatography-negative ion chemical ionization-mass spectrometry after derivatization with pentafluorobenzyl bromide. Ten healthy non-smoking males consumed identical amounts of broiled beef on five consecutive days. The morning after the first day of broiled beef consumption, urinary concentration of PhIP increased 14-38 fold above mean pre-feed concentration of PhIP in individual alkali-hydrolyzed urine samples. Following cessation of broiled beef consumption, urinary PhIP concentration declined to near pre-feed levels within 48-72 hrs. The ratio of total alkali-labile PhIP metabolites to unmetabolized PhIP varied by about 2.7-fold among subjects, ranging from 18:1 to 48:1, suggesting that interindividual differences in PhIP metabolism occur and can be detected by this method. This study of urinary PhIP following ingestion of meat cooked by charbroiling, that contains both HAs and polycyclic aromatic hydrocarbons, extends previous studies of ingestion of pan-fried meat that contains primarily HAs. The results indicate that significant amounts of PhIP are bioavailable from ingestion of charbroiled ground beef and that measurement of proportions of alkali-labile PhIP metabolites and parent PhIP in human urine may yield information on individual metabolism of ingested PhIP.

Chlorination of guanosine and other nucleosides by hypochlorous acid and myeloperoxidase of activated human neutrophils. Catalysis by nicotine and trimethylamine.

Activated human neutrophils secrete myeloperoxidase, which generates HOCl from H2O2 and Cl(-). We have found that various (2'-deoxy)nucleosides react with HOCl to form chlorinated (2'-deoxy)nucleosides, including novel 8-chloro(2'-deoxy)guanosine, 5-chloro(2'-deoxy)cytidine, and 8-chloro(2'-deoxy)adenosine formed in yields of 1.6, 1.6, and 0.2%, respectively, when 0.5 mM nucleoside reacted with 0.5 mM HOCl at pH 7.4. The relative chlorination, oxidation, and nitration activities of HOCl, myeloperoxidase, and activated human neutrophils in the presence and absence of nitrite were studied by analyzing 8-chloro-, 8-oxo-7,8-dihydro-, and 8-nitro-guanosine, respectively, using guanosine as a probe. 8-Chloroguanosine was always more easily formed than 8-oxo-7,8-dihydro- or 8-nitro-guanosine. Using electrospray ionization tandem mass spectrometry, we show that several chlorinated nucleosides including 8-chloro(2'-deoxy)guanosine are formed following exposure of isolated DNA or RNA to HOCl. Micromolar concentrations of tertiary amines such as nicotine and trimethylamine dramatically enhanced chlorination of free (2'-deoxy)nucleosides and nucleosides in RNA by HOCl. As the G-463A polymorphism of the MPO gene, which strongly reduces myeloperoxidase mRNA expression, is associated with a reduced risk of lung cancer, chlorination damage of DNA /RNA and nucleosides by myeloperoxidase and its enhancement by nicotine may be important in the pathophysiology of human diseases associated with tobacco habits.

Formation of spiroiminodihydantoin nucleoside by reaction of 8-oxo-7,8-dihydro-2'-deoxyguanosine with hypochlorous acid or a myeloperoxidase-H(2)O(2)-Cl(-) system.

Hypochlorous acid (HOCl), generated by myeloperoxidase from H(2)O(2) and Cl(-), is a strong chlorinating and oxidizing agent, playing an important role in host defense and inflammatory tissue injury. As several recent studies have shown that various oxidizing agents including peroxynitrite and singlet oxygen react readily with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) to yield further oxidized products, we have studied the reaction of 8-oxodGuo with reagent HOCl and with a myeloperoxidase-H(2)O(2)-Cl(-) system. When 1 mM 8-oxodGuo was reacted with 0.5 mM HOCl at pH 7.4 and 37 degrees C, two major products were formed. They were identified as the diastereomers of spiroiminodihydantoin deoxyribonucleoside (dSph) on the basis of their identical ESI-MS and UV spectra and HPLC retention times with those of the major reaction products which were reported to be formed in other oxidation systems including potassium monopersulfate plus cobalt (II) chloride, peroxynitrite plus thiol, and type II photosensitization. Under the above reaction conditions, the yield of the diastereomers of dSph was 0.38 mM, with 0.57 mM 8-oxodGuo remaining unreacted. Since the presence of 50% D(2)O, 10 mM sodium azide, or 2% ethanol did not affect the yield of the products, involvement of singlet oxygen and hydroxyl radical in the formation of dSph from 8-oxodGuo with HOCl was ruled out. A 1000-fold excess of dGuo did not inhibit the reaction of 8-oxodGuo with HOCl, indicating that 8-oxodGuo reacts more readily than dGuo with HOCl. dSph was also formed by reaction of 8-oxodGuo with myeloperoxidase in the presence of H(2)O(2) and Cl(-). Our results suggest that formation of dSph from 8-oxodGuo is mediated, possibly via an addition of Cl(+) to, or two-electron oxidation of 8-oxodGuo, with HOCl or the myeloperoxidase-H(2)O(2)-Cl(-) system.

Specific p53 mutations detected in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry.

Hepatocellular carcinoma (HCC), a common cause of cancer deaths worldwide, has several major etiological risk factors, including infection with the hepatitis viruses and exposure to aflatoxin B1. A specific missense mutation resulting from a guanine to thymine transversion at the third position of codon 249 in the p53 tumor suppressor gene has been reported in 10-70% of HCCs from areas of high dietary exposure to aflatoxin B1. Short oligonucleotide mass analysis was compared with DNA sequencing in 25 HCC samples for specific p53 mutations. Mutations were detected in 10 samples by short oligonucleotide mass analysis in agreement with DNA sequencing. Analysis of another 20 plasma and tumor pairs showed 11 tumors containing the specific mutation, and this change was detected in six of the paired plasma samples. Four of the plasma samples had detectable levels of the mutation; however, the tumors were negative, suggesting possible multiple independent HCCs. Ten plasma samples from healthy individuals were all negative. This molecular diagnostic technique has implications for prevention trials and for the early diagnosis of HCC.

Formation of N-nitrosamines and N-nitramines by the reaction of secondary amines with peroxynitrite and other reactive nitrogen species: comparison with nitrotyrosine formation.

Reactive nitrogen species, including nitrogen oxides (N(2)O(3) and N(2)O(4)), peroxynitrite (ONOO(-)), and nitryl chloride (NO(2)Cl), have been implicated as causes of inflammation and cancer. We studied reactions of secondary amines with peroxynitrite and found that both N-nitrosamines and N-nitramines were formed. Morpholine was more easily nitrosated by peroxynitrite at alkaline pH than at neutral pH, whereas its nitration by peroxynitrite was optimal at pH 8.5. The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at pH <7.5. For the morpholine-peroxynitrite reaction, nitration was enhanced by low concentrations of bicarbonate, but was inhibited by excess bicarbonate. Nitrosation was inhibited by excess bicarbonate. On this basis, we propose a free radical mechanism, involving one-electron oxidation by peroxynitrite of secondary amines to form amino radicals (R(2)N(*)), which react with nitric oxide ((*)NO) or nitrogen dioxide ((*)NO(2)) to yield nitroso and nitro secondary amines, respectively. Reaction of morpholine with NO(*) and superoxide anion (O(2)(*)(-)), which were concomitantly produced from spermine NONOate and by the xanthine oxidase systems, respectively, also yielded nitromorpholine, but its yield was <1% of that of nitrosomorpholine. NO(*) alone increased the extent of nitrosomorpholine formation in a dose-dependent manner, and concomitant production of O(2)(*)(-) inhibited its formation. Reactions of morpholine with nitrite plus HOCl or nitrite plus H(2)O(2), with or without addition of myeloperoxidase or horseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants. Tyrosine was nitrated easily by synthetic peroxynitrite, by NaNO(2) plus H(2)O(2) with myeloperoxidase, and by NaNO(2) plus H(2)O(2) under acidic conditions. Nitrated secondary amines, e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated by reactive nitrogen species.

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.

Genotyping by mass spectrometric analysis of short DNA fragments.

A method has been developed to produce small DNA fragments from PCR products for analysis of defined DNA variations by mass spectrometry. The genomic region to be analyzed is PCR-amplified with primers containing a sequence for the type IIS restriction endonuclease Bpml. Bpml digestion of the resultant PCR products yields fragments as small as seven bases, which are then analyzed by electrospray ionization mass spectrometry. The approach was validated using seven different variants within the APC tumor suppressor gene, in which a perfect correlation was obtained with DNA sequencing. Both the sense and antisense strands were analyzed independently, and several variants can be analyzed simultaneously. These results provide the basis for a generally applicable and highly accurate method that directly queries the mass of variant DNA sequences.

4-Hydroxy-1-(3-pyridyl)-1-butanone-hemoglobin adducts as biomarkers of exposure to tobacco smoke: validation of a method to be used in multicenter studies.

Hemoglobin (Hb) adducts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), a metabolite of two tobacco-specific nitrosamines [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine], were measured as biomarkers of exposure to tobacco smoke as part of a study on genetic alterations and susceptibility to lung cancer among nonsmokers. HPB-Hb adducts were measured after collection of RBCs by Ficoll gradient in six collaborating centers, release of HPB by alkaline hydrolysis from Hb, clean-up by solid-phase extraction, and analysis of an electron-capturing derivative by gas chromatography-electron capture mass spectrometry. Prior to analysis of samples from study subjects, the reproducibility of this approach was validated in blood from donors. The coefficient of variation of reproducibility of paired aliquots from five samples ranged from 7 to 25%; the within-sample reproducibilities of four and eight aliquots were 4 and 16%, respectively. The study subjects consisted of 18 smokers and 52 never-smokers. HPB-Hb adduct levels were significantly higher (P = 0.02) in smokers (26 +/- 13 fmol HPB/g Hb) than in never-smokers (20 +/- 8 fmol HPB/g Hb). There was no difference between sexes. These results suggest that the level of HPB-Hb adducts, measured using a method modified to facilitate use in multicenter studies, can be a useful biomarker of exposure to tobacco smoke.

DNA adducts of heterocyclic amines: formation, removal and inhibition by dietary components.

The dietary mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic in rodents. In F344 rats PhIP induces mammary tumors in females and colon tumors in males, while IQ induces tumors principally in the liver, Zymbal gland and intestines. In CDF1 mice, IQ induces liver, lung and forestomach tumors. We have evaluated the dynamics of formation, removal and inhibition of PhIP- and IQ-DNA adducts in these rodents. After bolus doses (50 mg/kg, by gavage) of IQ or PhIP, both IQ- and PhIP-DNA adducts were removed rapidly from both target and nontarget organs, while after 3-4 weeks of feeding IQ or PhIP (0.01-0.04%) adduct removal was much slower. Gavaging of male F344 rats with PhIP (0.1-1000 micrograms/kg/day) for 23 days resulted in accumulation of PhIP-DNA adducts in various organs, but adducts were detectable only at 100 or 1000 micrograms/kg/day. Urinary excretion of unchanged PhIP was a constant proportion (1.6-2.1%) of the daily dose over the entire dose range and was independent of duration of exposure. When weanling female F344 rats were exposed to dietary PhIP (0.01-0.04%) for 1-4 weeks, the presence of either conjugated linoleic acid (CLA; 0.1-1.0%) or indole-3-carbinol (13C; 0.1%) in the diet inhibited PhIP-DNA adduct formation (58-99%) in various organs, including the mammary gland and the colon. Similarly, the inclusion of 0.075% 4-ipomeanol (IPO) in the diet of male CDF1 mice exposed for 3 weeks to dietary IQ (0.01%) resulted in inhibition of IQ-DNA adduct formation (30-59%) in the target organs (liver, lungs, stomach) but not in a number of other organs. It is concluded that (1) the rate of PhIP- and IQ-DNA adduct removal depends on the dose and frequency of administration, (2) urinary PhIP may be a good biomarker of recent PhIP exposure and (3) CLA, I3C and IPO are potential chemopreventive agents against PhIP- or IQ-induced tumors in rodents.

Markers of internal dose: chemical agents.

Biomarkers of internal dose measure the level of a carcinogen or one of its metabolites in a tissue or a body fluid such as urine or blood. The choice of a biomarker of internal dose for a particular epidemiological study or type of study requires careful consideration of the period of exposure to which the biomarker relates, host factors related to carcinogen metabolism, invasiveness of sampling, reliability and cost of the biomarker. Before a new biomarker is adopted, it is important to assess these characteristics in transitional studies to ensure that the biomarker will be applied appropriately. Biomarkers of internal dose have been applied most successfully in ecological studies and nested case-control studies, and are especially useful when they provide information about long-term carcinogen exposure.

Effects of phenethyl isothiocyanate on metabolism and on genotoxicity of dimethylnitrosamine and 2-amino-1-methyl-6-phenylimidazo[4, 5-beta]pyridine (PhIP).

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, inhibited the genotoxic effects of N-nitrosodimethylamine (DMN) and of 2-amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine (PhIP) in differential DNA repair assays with E. coli K-12 strains in vitro and in animal mediated assays with mice. In Salmonella typhimurium, the mutagenic activities of DMN and PhIP measured after activation with S-9 homogenates from several organs of PEITC-treated mice were substantially lower than those obtained with homogenates of untreated animals as well. PEITC also reduced the formation of micronuclei by DMN in metabolically competent Hep-G-2 cells of human origin but was ineffective in combination with PhIP. Biochemical investigations showed that the prevention of genotoxic effects of DMN by PEITC results form an inhibition of its alpha-hydroxylation. The effect of oral administration of PEITC on the formation of DNA adducts of PhIP was examined in the colon and liver of mice. No inhibition of adduct formation was observed in these experiments. Biochemical experiments showed that PEITC reduces not only the metabolic activation of PhIP via 2-hydroxyamino PhIP but also inhibits a detoxification pathway (formation of 4-hydroxy PhIP). The present results can be taken as an indication that the anticarcinogenic activities of isothiocyanates towards nitrosamines are paralleled by antimutagenic effects, and that probably no such protective effects occur in combination with heterocyclic amines. Furthermore, our findings show that the effects of chemopreventive agents demonstrated in bacteria in vitro cannot always be extrapolated to reactions occurring in intact mammalian cells.

Validation in rats of two biomarkers of exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): PhIP-DNA adducts and urinary PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts in white blood cells and tissues and unmetabolized PhIP in urine were validated as biomarkers of exposure in male Fischer-344 rats treated with daily PhIP doses ranging from 1 to 0.0001 mg/kg. At the end of the 23 day treatment period all rats were killed and their blood and 10 tissues were collected for isolation of DNA and analysis of PhIP-DNA adducts by 32P-postlabeling and alkaline hydrolysis with GC/MS. PhIP-DNA adducts could be detected only in animals receiving 1 or 0.1 mg/kg/day, with highest adduct levels in the pancreas, heart and kidneys. There was a good correlation (r = 0.77, P < 0.005) between the two methods of analysis, with average adduct levels determined by 32P-postlabeling approximately 1.4 times higher than those determined by alkaline hydrolysis with GC/MS. PhIP-DNA adducts accumulated in most tissues, especially in the liver, kidneys, heart and pancreas, with lower levels in the white blood cells, small intestine, stomach, colon and cecum. Using GC/MS levels of unmetabolized PhIP were measurable in four weekly 24 h urine samples even at 0.0001 mg/kg/day, a dose comparable with reported human dietary exposure. A linear dose-response was obtained for excretion of unmetabolized PhIP across the range of doses, with approximately 1.8% of the dose excreted daily, largely independent of the number of doses. No PhIP was detected in the urine of untreated rats. If it can be shown that a constant percentage of PhIP is excreted unchanged in human urine, irrespective of dose, as has been found with the rat, measurement of urinary PhIP could be used as an accurate measure of dietary exposure to this amine in man.

Formation of 8-nitroguanine by the reaction of guanine with peroxynitrite in vitro.

Nitric oxide and superoxide anion, both formed in inflamed tissues, react rapidly to form the peroxynitrite anion (ONOO-), a strong oxidant which can initiate reactions characteristic of hydroxyl radical (HO.), nitronium ion (NO2+) and nitrogen dioxide radical (NO2.). Peroxynitrite, therefore, may cause DNA or tissue damage, contributing to the multistage carcinogenesis process. We have studied reactions of various bases, nucleosides or deoxynucleosides with peroxynitrite in vitro. Guanine reacted rapidly with peroxynitrite under physiological conditions and formed several substances, two of which were yellow, a characteristic of nitro and nitroso compounds. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for approximately 80% of all compounds formed) as 8-nitroguanine. Its formation was maximal at approximately pH 8 and increased dose-dependently with peroxynitrite concentration, but was not dependent on guanine concentration. The presence of ferric ions, which has been shown to catalyse nitration of tyrosine, did not affect nitration of guanine. 8-Nitroguanine could act as a specific marker for DNA damage induced by peroxynitrite in inflamed tissues.

Ortho- and meta-tyrosine formation from phenylalanine in human saliva as a marker of hydroxyl radical generation during betel quid chewing.

The habit of betel quid chewing, common in South-East Asia and the South Pacific islands, is causally associated with an increased risk of oral cancer. Reactive oxygen species formed from polyphenolic betel quid ingredients and lime at alkaline pH have been implicated as the agents responsible for DNA and tissue damage. To determine whether hydroxyl radical (HO.) is generated in the human oral cavity during chewing of betel quid, the formation of o- and m-tyrosine from L-phenylalanine was measured. Both o- and m-tyrosine were formed in vitro in the presence of extracts of areca nut and/or catechu, transition metal ions such as Cu2+ and Fe2+ and lime or sodium carbonate (alkaline pH). Omission of any of these ingredients from the reaction mixture significantly reduced the yield of tyrosines. Hydroxyl radical scavengers such as ethanol, D-mannitol and dimethylsulfoxide inhibited the phenylalanine oxidation in a dose-dependent fashion. Five volunteers chewed betel quid consisting of betel leaf, areca nut, catechu and slaked lime (without tobacco). Their saliva, collected after chewing betel quid, contained high concentrations of p-tyrosine, but no appreciable amounts of o- or m-tyrosine. Saliva samples from the same subjects after chewing betel quid to which 20 mg phenylalanine had been added contained o- and m-tyrosine at concentrations ranging from 1010 to 3000 nM and from 1110 to 3140 nM respectively. These levels were significantly higher (P < 0.005) than those of subjects who kept phenylalanine in the oral cavity without betel quid, which ranged from 14 to 70 nM for o-tyrosine and from 10 to 35 nM for m-tyrosine. These studies clearly demonstrate that the HO. radical is formed in the human oral cavity during betel quid chewing and is probably implicated in the genetic damage that has been observed in oral epithelial cells of chewers.

Metabolic activation and DNA adduct detection of PhIP in dogs, rats, and humans in relation to urinary bladder and colon carcinogenesis.

The metabolic activation of the heterocyclic amine carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was examined in dogs and rats as models for urinary bladder and colon carcinogenesis, respectively. The results indicate that unconjugated N-OH-PhIP is not excreted in the urine after oral dosing with PhIP and that the two isomeric N-glucuronides of N-OH-PhIP, which are formed as major metabolites, are stable under acidic conditions. These data suggest that PhIP is unlikely to serve as a urinary bladder carcinogen in either species. Using metabolic inhibitors, bile duct ligation, and intravenous dosing studies, a new hypothesis for colorectal carcinogenesis is proposed involving N-oxidation of PhIP by hepatic cytochrome P-4501A2 (CYP1A2) and O-acetylation by the polymorphic acetyltransferase (NAT2). The resulting N-hydroxy and N-acetoxy metabolites both appear to be transported through the circulation to the colon mucosa, forming covalent DNA adducts. Glucuronidation and reaction with glutathione appear to serve as detoxification pathways. In humans, individuals who are phenotypically rapid metabolizers for both CYP1A2 and NAT2 are significantly higher (p = 0.0015) in colorectal cancer/poly cases vs. controls; and PhIP-DNA adducts can be detected in human colon samples. These studies provide strong evidence that PhIP and other heterocyclic amines play an important role in the etiology of human colorectal cancer.

Analysis of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine in rat and human tissues by alkaline hydrolysis and gas chromatography/electron capture mass spectrometry: validation by comparison with 32P-postlabeling.

A sensitive and specific method has been developed to measure levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) adducted to DNA in tissues. The method is based on alkaline hydrolysis of PhIP from DNA, followed by organic solvent extraction, derivatization to form the electron-capturing bis(pentafluorobenzyl) derivative, and analysis by gas chromatography/electron capture mass spectrometry (GC/MS) using a deuterium-labeled internal standard. The method can detect PhIP-DNA adducts at levels down to 0.03 fmol of PhIP/micrograms of DNA (1 PhIP adduct/10(8) normal nucleotides) for a 100 micrograms sample of DNA. The method is reproducible for sample sizes ranging up to at least 1000 micrograms of DNA. A series of 20 DNA samples from 5 tissues of rats treated with a single oral dose of PhIP were analyzed both by alkaline hydrolysis-GC/MS and by 32P-postlabeling. Results from the two methods were highly correlated (r2 = 0.83), with adduct levels determined by alkaline hydrolysis-GC/MS averaging about 60% of the levels determined by 32P-postlabeling. A pilot survey of 24 individual human tissue DNA samples, including pancreas (n = 12), colon mucosa (n = 6), and urinary bladder epithelium (n = 6), was carried out by alkaline hydrolysis-GC/MS and 32P-postlabeling. Both methods provided evidence for PhIP-DNA adducts in two of the colon samples, but not in the samples from human pancreas or urinary bladder.

Noninvasive methods for measuring DNA alkylation in experimental animals and humans.

Alkylpurines are liberated from alkylated DNA by glycosylase repair enzymes and, in most cases, excreted in urine without further metabolism. This phenomenon forms the basis of noninvasive methods to measure DNA alkylation in vivo. In the case of methyl adducts, such as 7-methylguanine (7-MeGua), natural backgrounds exist due to RNA turnover. However, deuterated (d3) methylating agents or precursors give rise to d3-7-MeGua and d3-3-methyladenine (3-MeAde), which can be readily quantitated using gas chromatography-mass spectrometry (GC-MS). A deuterated probe drug, such as d6-aminopyrine, can be used to measure endogenous nitrosation levels in experimental animals. In contrast, for higher alkyl homologues of alkylpurines, natural backgrounds are low or nonexistent and can be directly measured by GC-MS using stable isotope labeled internal standards. For example, increased levels of urinary 3-ethyladenine were observed in cigarette smokers. Due to recent advances in analytical methodology, notably immunoaffinity cleanup of urine, measurements of excreted DNA adducts can be used in studies in human populations exposed to low levels of alkylating carcinogens.

Urinary markers for measuring exposure to endogenous and exogenous alkylating agents and precursors.

Noninvasive methodologies for measuring carcinogen exposure in humans, based on the use of urinary markers, are being developed and validated for use in molecular epidemiological studies. A range of 3-alkyladenines can be determined in urine samples by an immunoaffinity purification-GC/MS approach [3-methyladenine, 3-ethyladenine, 3-(2-hydroxyethyl)adenine, and 3-benzyladenine]. Using this method, recent results in human subjects suggest that urinary 3-alkyladenines are potentially useful markers of alkylating agent exposure, particularly where the backgrounds of such adducts are much lower than 3-methyladenine. Urinary excretion of S-benzylmercapturic acid has been studied in experimental animals as a marker of exposure to benzylating agents such as N-nitroso-methylbenzylamine. 3-Nitrotyrosine (NTyr) is formed in vivo in tissue or blood proteins after exposure to nitrosating and/or nitrating agents such as tetranitromethane. After turnover of proteins, NTyr is released and excreted in urine as metabolites 3-nitro-4-hydroxy-phenylacetic acid and 3-nitro-4-hydroxyphenylacetic acid, which are determined by GC with a thermal energy analyzer. The sensitivity and specificity, combined with ease of use, of these noninvasive biomonitoring approaches means that they may be readily incorporated into molecular epidemiological studies in which exposure to nitrosating and alkylating agents may be important risk factors.

Gas chromatography-mass spectrometry analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and feces.

A method has been developed to measure levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) excreted in urine and feces. The method involves organic solvent extraction, derivatization to form electron-capturing bis-pentafluorobenzyl derivatives, and analysis by gas chromatography-negative ion chemical ionization mass spectrometry using a deuterium-labeled internal standard. The method can detect PhIP at levels of less than 1 ng/g in rat urine (5 ng/24 hr) and 5 ng/g (wet weight) in rat feces (50 ng/24 hr). Sprague-Dawley rats given a single 50 micrograms dose of PhIP by gavage excreted an average of 0.6% of the dose in the urine and 25% of the dose in the feces as unchanged PhIP, in the first 4 days after treatment. To make this method applicable for the analyses of biological fluids of PhIP-exposed human subjects, it is now being improved by using immunoaffinity chromatography.