Analysis of 4-aminobiphenyl hemoglobin adducts in smokers and nonsmokers by pseudo capillary on-column gas chromatography- tandem mass spectrometry.

We describe here a hemoglobin adduct assay applied to an analysis of samples from smokers and nonsmokers. The assay includes a sensitive method for quantification of orthotoluidine 2-aminonaphthylene, and 3- and 4-aminobiphenyl hemoglobin adducts in human blood using capillary gas chromatography-tandem mass spectrometry. Basic hydrolysis and derivatization with pentafluoropropionic acid anhydride are followed by programmable temperature vaporization and pseudo on-column capillary gas chromatography with positive electron ionization tandem mass spectrometry analysis. Standard deviation of calibration curves (n = 6) shows that the limits of detection for o-toluidine, 2-aminonaphthylene, and 3- and 4-aminobiphenyl were 0.23, 0.39, 0.30, and 0.24 pg total on-column, respectively. The effective working limit of detection is estimated at approximately 5.22 pg/g Hb and 18.73 pg/g Hb for 4-aminobiphenyl and 2-aminonaphthylene, respectively. In a group that was predominately male and African-American, the level of 4-aminobiphenyl Hb adducts was significantly different between smokers and nonsmokers. Among 93 nonsmokers with serum cotinine concentrations less than 10 ng/mL, the geometric mean (95% CI) concentration of 4-aminobiphenyl was 29.9 pg/g hemoglobin (Hb; 29.4 to 30.4). Conversely, in 100 smokers the 4-aminobiphenyl adducts geometric mean concentration was significantly greater at 73.0 pg/g Hb (72.6 to 73.4). 4-Aminobiphenyl hemoglobin adduct and serum cotinine concentrations were correlated (r = 0.496; p < 0.0001; n = 193). In 15% of smokers, 3-aminobiphenyl was detected at low concentration. Adduct levels of 2-aminonaphthylene and ortho-toluidine were not significantly different between the smoker and nonsmoker participants. Our study shows that 4-aminobiphenyl Hb adducts remain the preferred biomarker for identifying people exposed to aromatic amines from tobacco smoke.

Secondhand smoking, 4-aminobiphenyl, and bladder cancer: two meta-analyses.

OBJECTIVE: To quantify the relation between secondhand smoking (SHS) and levels of 4-aminobiphenyl (4-ABP; in urine or blood) and SHS and bladder cancer risk in nonsmokers. METHODS: PubMed and Embase were searched (search terms to represent SHS, bladder cancer, and 4-ABP) to conduct two meta-analyses. Information about gender and age of participants, mean 4-ABP level for each SHS category, number of subjects, relative risk or odds ratio and 95% confidence intervals (95% CI) in each SHS category, and covariates for which adjustment was made was extracted based on predefined inclusion and exclusion criteria. Random-effects analyses were done using STATA (version 9). RESULTS: A 118 studies were reviewed for information on SHS and 4-ABP (31 studies) and SHS and bladder cancer risk (87 studies). Of those, seven case-control studies were included for analysis of SHS and 4-ABP and eight articles (three cohort and five case-control studies) for SHS and bladder cancer risk. A random-effects model found a pooled standardized mean difference of 1.47 (95% CI, 0.23-2.71), indicating higher levels of 4-ABP among nonsmokers exposed to SHS. A random-effects model showed no evidence for an association between SHS and bladder cancer risk (relative risk, 0.99; 95% CI, 0.86-1.14), comparing nonsmokers with and without SHS exposure. CONCLUSION: Higher levels of 4-ABP were significantly associated with SHS exposure, which is consistent with earlier findings for 4-ABP levels in sidestream smoke. The current evidence indicates that there is no association between SHS and bladder cancer, but future studies that address methodologic limitations are needed to further clarify this important question.

Hemoglobin adducts of the human bladder carcinogen o-toluidine after treatment with the local anesthetic prilocaine.

Prilocaine, a widely used local anesthetic, is metabolized to o-toluidine which is classified as human carcinogen. We aimed to assess the impact of prilocaine-treatment on hemoglobin adducts from o-toluidine. Blood samples were obtained before and 24h after receiving prilocaine local anesthesia (Xylonest, 100mg) from 20 head and neck surgery patients and 6 healthy volunteers. Hemoglobin adducts of o-toluidine and 4-aminobiphenyl were determined by gas chromatography/mass spectrometry. Hemoglobin adducts of o-toluidine were significantly increased 24h after 100mg prilocaine-treatment by 21.6+/-12.8ng/g hemoglobin (mean+/-S.D., N=26; P<0.0001). This corresponds to a 6-360-fold increase of o-toluidine adduct levels in 25 patients from 0.54+/-0.95ng/g before treatment to 22.0+/-13.2ng/g 24h after surgery (mean+/-S.D.). Because of an extremely high background level the increase was only 1.6-fold in one patient (40.9ng/g before and 64.4ng/g 24h after prilocaine injection). Current smoking had no influence on background values and on the increase of o-toluidine adducts. No treatment-related differences were seen in mean hemoglobin adduct levels of 4-aminobiphenyl which were significantly higher in smokers, 0.149+/-0.096ng/g (mean+/-S.D., N=8) as compared to nonsmokers 0.036+/-0.035ng/g (mean+/-S.D., N=16; P<0.01). In conclusion, prilocaine anesthesia leads to a massive increase of hemoglobin adducts of the carcinogenic arylamine o-toluidine. This implies a carcinogenic risk which should be taken into account in preventive hazard minimization.

Effect of diet on haemoglobin adducts from 4-aminobiphenyl in rats.

High levels of haemoglobin (Hb) adducts from 4-aminobiphenyl (4-ABP), a proven human carcinogen, have been reported in untreated animals from different laboratories fed various commercial standard diets. Therefore, the impact of dietary modifications on 4-ABP Hb adducts was investigated. Female Sprague-Dawley rats were fed a regular standard diet or three different test diets for 4 weeks. 4-ABP Hb adducts were significantly lower in rats on vegetable-based test diets #2 (596+/-183 pg/g Hb, P = 0.028) and #3 (537+/-48 pg/g Hb, P = 0.009) compared with controls (974+/-154 pg/g Hb). Cereal-based test diet #1 (1080+/-388 pg/g Hb) had no influence on the basal Hb adduct levels determined before the start of the experiment (1054+/-163 pg/g Hb). In conclusion, the body burden of rats with 4-ABP could be significantly reduced by dietary modifications.

N-acetyltransferase 2 phenotype but not NAT1*10 genotype affects aminobiphenyl-hemoglobin adduct levels.

Aminobiphenyls (ABPs) in tobacco have been implicated in bladder cancer etiology in smokers. N-Acetylation of ABPs in the liver, predominantly by the N-acetyltransferase 2 (NAT2) isozyme, represents a detoxification pathway, whereas O-acetylation of N-hydroxy-ABPs in the bladder, predominantly by the N-acetyltransferase 1 (NAT1) isozyme, represents a bioactivation pathway. We and others have demonstrated that NAT2 phenotype affects 3- and 4-ABP-hemoglobin adduct levels (higher levels in slow acetylators), which are considered valid biomarkers of the internal dose of ABP to the bladder. We have also shown that NAT1 genotype (NAT1*10 allele) is associated with increased DNA adduct levels in urothelial tissue and higher risk of bladder cancer among smokers. It is not known whether NAT1*10 genotype influences ABP-hemoglobin adduct levels. Therefore, we assessed 403 primarily non-Hispanic white residents of Los Angeles County for their NAT2 acetylator phenotype, NAT1*10 acetylator genotype, and 3- and 4-ABP-hemoglobin adduct levels. Eighty-two subjects were current tobacco smokers of varying intensities. Tobacco smokers had significantly higher mean 3- and 4-ABP-hemoglobin adduct levels relative to nonsmokers. The levels increased with increased amounts smoked per day (two-sided, P < 0.0001 in all cases). With adjustment for NAT1 genotype and race, the smoking-adjusted geometric mean level of 3-ABP-hemoglobin adducts in NAT2 slow acetylators was 47% higher than that in NAT2 rapid acetylators (P = 0.01). The comparable value for 4-ABP-hemoglobin adducts was 17% (P = 0.02). In contrast, no association between NAT1*10 genotype and 3- or 4 ABP-hemoglobin adduct levels was observed after adjustment for NAT2 phenotype, smoking, and race. The present study suggests that the impact of the NAT1*10 genotype on 3- and 4-ABP-hemoglobin adducts is noninformative on the possible association between NAT1 activity and bladder cancer risk.

Molecular and genetic damage from environmental tobacco smoke in young children.

To assess the risks of early life exposure to environmental tobacco smoke (ETS), we tested whether four biomarkers in peripheral blood were associated with home ETS exposure in Hispanic and African-American children. The biomarkers included cotinine (a metabolite of nicotine) and three indicators of molecular and genetic damage from mutagens/carcinogens, protein adducts formed by the carcinogens 4-aminobiphenyl (4-ABP) and polycyclic aromatic hydrocarbons (PAHs), and sister chromatid exchanges (SCEs). We also explored possible ethnic differences in biomarkers. The study cohort comprised 109 Hispanic and African-American preschool children (1-6 years of age). Plasma cotinine was analyzed by gas chromatography, 4-ABP-hemoglobin adducts by gas chromatography-mass spectroscopy, PAH-albumin adducts by ELISA, and SCEs by cytogenetic techniques. Data on the amount of smoking by mothers (average 10.5 cigarettes per day) and other household members and regular visitors (average 6.5 cigarettes per day) were obtained by interview-administered questionnaires. Cotinine, 4-ABP-hemoglobin adducts, and PAH-albumin were significantly higher (P < 0.05) in the ETS-exposed children compared with the unexposed. SCEs were marginally higher (P = 0.076). African-American children had higher levels of cotinine (P = 0.059) and PAH-albumin (P = 0.02) than Hispanic children, after controlling for exposure to ETS. These results indicate molecular and genetic damage in minority children with

The use of 4-aminobiphenyl hemoglobin adducts and aromatic DNA adducts in lymphocytes of smokers as biomarkers of exposure.

Two biomarkers of exposure to cigarette smoke, 4-aminobiphenyl-hemoglobin (Hb) adducts and aromatic DNA adducts in lymphocytes, were determined from a population of 55 smokers and 4 nonsmokers. The levels of these adducts were related to daily cigarette consumption and also to (calculated) tar and nicotine intake. The Hb adduct levels seemed to correspond best to the number of cigarettes (cig) smoked, but at a cigarette consumption of >30 cig/day, a saturation effect was observed. Lymphocytic DNA adducts also correlated well with cigarette and tar consumption; for this type of adduct, a saturation level was reached at a dose of approximately 15-20 cig/day. From a subpopulation, a second sample was obtained after 2 months, and the adduct levels were compared with their initial adduct levels. Strong correlations were found between the first and second DNA adduct measurements (r = 0.84). In another subpopulation, resampling was performed after 6 months. No correlation between DNA adduct levels in the first and last samples was found, but 4-aminobiphenyl Hb adduct levels were strongly correlated (r = 0.78), the absolute quantities measured being comparable (paired t test: t = -1.27, P = 0.22, n = 15). We found no influence of GSTM1 and NAT2 polymorphisms on Hb adduct formation and of GSTM1 polymorphism on aromatic DNA adduct formation. A significantly lower aromatic DNA adduct level was observed for intermediate acetylators when compared to slow acetylators.

Characterization of 4-aminobiphenyl-hemoglobin adducts in maternal and fetal blood-samples.

The maternal-fetal exchange of the potent tobacco-related human carcinogen 4-aminobiphenyl was studied in women smokers during pregnancy. The number of cigarettes smoked per day by each of the women in the study was assessed via questionnaire and by measurement by immunoassay of serum and urine cotinine in maternal and fetal blood samples. Maternal and fetal blood samples were classified as coming from nonsmokers (n = 74), individuals smoking less than 1 pack of cigarettes per day (n = 16), individuals smoking 1 pack of cigarettes per day (n = 19), individuals smoking 1-2 packs of cigarettes per day (n = 19), and individuals smoking greater than 2 packs of cigarettes per day (n = 20). Both maternal and fetal blood samples were obtained at the time of delivery. 4-Aminobiphenyl was extracted from both maternal and fetal blood samples using organic extractions and the released amine was qualitatively and quantitatively characterized by analysis of the samples by gas chromatographic and mass spectrometric analysis. Background levels of 4-aminobiphenyl-hemoglobin adducts were detected in maternal nonsmokers (18.3 +/- 12.7 pg 4-aminobiphenyl/g hemoglobin, mean +/- SD) and in fetal samples (8.88 +/- 5.8 pg/g hemoglobin). Increasing levels of 4-aminobiphenyl-hemoglobin adducts were found as the smoking status of the women increased, ranging from 144 +/- 22.2 ( < 1 pack/d) to 633 +/- 87.9 ( > 2 packs/d). A corresponding increase in the presence of fetal 4-aminobiphenyl-hemoglobin adducts was also detected (74 +/- 17.8, < 1 pack/d, to 319 +/- 50.5, > 2 packs/d). This study confirms that the potent tobacco-related carcinogen 4-aminobiphenyl crosses the human placenta and binds to fetal hemoglobin in significantly higher concentrations in smokers when compared to nonsmokers.

HPLC and GC/MS determination of 4-aminobiphenyl haemoglobin adducts in fetuses exposed to the tobacco smoke carcinogen in utero.

Maternal-fetal exchange of the potent tobacco-related human carcinogen, 4-aminobiphenyl, was studied in women nonsmokers and in women smokers as well as in the corresponding fetuses during pregnancy. Smoking status of the women in the study was assessed via questionnaire and measurement by immunoassay of serum cotinine in maternal and fetal blood samples. 4-Aminobiphenyl was extracted from both maternal and fetal blood samples using organic solvent extractions and the released amine was qualitatively and quantitatively characterized by analysis of the samples by high pressure liquid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (GC/MS). Background levels (pg 4-aminobiphenyl/g haemoglobin) of 4-aminobiphenyl-haemoglobin adducts were detected in maternal smokers (mean +/- S.D; 29.6 +/- 16.2 (GC/MS); 23.7 +/- 13.5 (HPLC) and in fetal samples (14.0 +/- 6.5 (GCMS); 10.0 +/- 4.6 (HPLC). Elevated levels of 4-aminobiphenyl-haemoglobin adducts were found in maternal smokers (488 +/- 174 (GC/MS); 423 +/- 154 (HPLC). as well as in the corresponding fetal blood samples (244 +/- 91 (GC/MS); 197 +/- 77 (HPLC). This study confirms that a potent tobacco-related carcinogen, 4-aminobiphenyl, crosses the human placenta and binds to fetal haemoglobin in significantly higher concentrations in smokers when compared to nonsmokers.

Tobacco-specific nitrosamines--metabolism and biological monitoring of exposure to tobacco products.

Tobacco-specific nitrosamines are derived from nicotine and related tobacco alkaloids and can be detected in tobacco products as well as in mainstream and sidestream smoke. Two of them, N-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, are strong carcinogens in laboratory animals. Because of its organospecificity for the lung, the latter is considered to be a causative factor in tobacco-related human lung cancer. Upon metabolic activation both nitrosamines give rise to a common reactive intermediate binding to macromolecules such as DNA and haemoglobin and hydrolysing to 4-hydroxy-1-(3-pyridyl)-1-butanone. Because of easy access to large quantities of haemoglobin from blood samples, it is most suitable for biomonitoring human exposure to tobacco-specific nitrosamines. A highly sensitive analytical method for determination of femtogram amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone provides an approach to assess individual exposure to active and passive smoking.

Frequency of urination and its effects on metabolism, pharmacokinetics, blood hemoglobin adduct formation, and liver and urinary bladder DNA adduct levels in beagle dogs given the carcinogen 4-aminobiphenyl.

The human urinary bladder carcinogen, 4-aminobiphenyl (ABP), is known to undergo hepatic metabolism to an N-hydroxy arylamine and its corresponding N-glucuronide. It has been proposed that these metabolites are both transported through the blood via renal filtration to the urinary bladder lumen where acidic pH can facilitate the hydrolysis of the N-glucuronide and enhance the conversion of N-hydroxy-4-aminobiphenyl (N-OH-ABP) to a reactive electrophile that will form covalent adducts with urothelial DNA. Blood ABP-hemoglobin adducts, which have been used to monitor human exposure to ABP, are believed to be formed by reactions within the erythrocyte involving N-OH-ABP that has entered the circulation from the liver or from reabsorption across the urothelium. To test these hypotheses directly, experimental data were obtained from female beagles given [3H]ABP (p.o., i.v., or intraurethrally). [3H]N-OH-ABP (i.v. or intraurethrally), or [3H]N-OH-ABP N-glucuronide (i.v.). Analyses included determinations of total ABP in whole blood and plasma, ABP-hemoglobin adducts in blood erythrocytes, ABP and N-OH-ABP levels (free and N-glucuronide) in urine, urine pH, frequency of urination (controlled by urethral catheter), rates of reabsorption of ABP and N-OH-ABP across the urothelium, and apparent volumes of distribution in the blood/tissue compartment. The major ABP-DNA adduct, N-(guan-8-yl)-4-aminobiphenyl, was also measured in urothelial and liver DNA using a sensitive immunochemical method. An analog/digital hybrid computer was then utilized to construct a multicompartmental pharmacokinetic model for ABP and its metabolites that separates: (a) absorption; (b) hepatic metabolism and distribution in blood and tissues; (c) ABP-hemoglobin adduct formation; (d) hydrolysis and reabsorption in the urinary bladder lumen; and (e) excretion. Using this model, cumulative exposure of the urothelium to free N-OH-ABP was simulated from the experimental data and used to predict ABP-DNA adduct formation in the urothelium. The results indicated that exposure to N-OH-ABP and subsequent ABP-DNA adduct formation are directly dependent on voiding frequency and to a lesser extent on urine pH. This was primarily due to the finding that, after p.o. dosing of ABP to dogs, the major portion of the total N-OH-ABP entering the bladder lumen was free N-OH-ABP (0.7% of the dose), with much lower amounts as the acid-labile N-glucuronide (0.3% of the dose).(ABSTRACT TRUNCATED AT 400 WORDS)

The metabolism of 4-aminobiphenyl in rat. II. Reaction of N-hydroxy-4-aminobiphenyl with rat blood in vitro.

1. N-Hydroxy-4-aminobiphenyl (N-hydroxy-ABP) reacts with HbFe2+ of rat blood in vitro at a molar ratio of 1:47 to produce 20% HbFe3+ within 1 min; N-hydroxy-ABP oxidized 9.4 equiv. of HbFe2+. N-hydroxy-ABP rapidly disappeared and HbFe3+ was reduced at a rate of 44 microM/min. 2. On titration of rat blood in vitro with N-hydroxy-ABP up to 0.81 mM, 4-nitrosobiphenyl (nitroso-BP) disappeared within 5 min; with concn of N-hydroxy-ABP greater than 0.81 mM, N-hydroxy-ABP was present also as nitroso-BP, indicating saturation of reactive binding sites. When N-hydroxy-ABP reacted with HbFe2+ at a molar ratio of 1:103 to 1:1.9, 13 to 1.3 equiv. of HbFe3+ were formed per mol of N-hydroxy-ABP in 5 min, indicating that with increasing N-hydroxy-ABP concn side-reactions increased. 3. After incubation of N-hydroxy-ABP (1.72 mM) with rat Hb (7.66 mM HbFe2+), nitroso-BP disappeared with a half-life of 1 min, maximal HbFe3+ of 72% occurred at 47 min, and the concn of 4-aminobiphenyl (ABP) increased at a rate of 51 nmol/ml per h. 4. In rats injected with 0.24 mmol/kg ABP, HbFe3+ concn plateaued at 56% after 75 min, indicating an equilibrium between HbFe3+ formation and HbFe3+ reduction. Such equilibrium was simulated by titrating rat blood in vitro with N-hydroxy-ABP for 1 h. 5. The long-lasting HbFe3+ formation by ABP in rat results from a cycle of activation of ABP to N-hydroxy-ABP, its rapid co-oxidation with HbFe2+ to form HbFe3+ and nitroso-BP, and binding of nitroso-BP to erythrocyte thiol groups. ABP is released from the Hb adduct and enters a new cycle of activation and inactivation, until terminated by ring-hydroxylation.

The metabolism of 4-aminobiphenyl in rat. I. Reaction of N-hydroxy-4-aminobiphenyl with rat blood in vivo.

1. 3H-4-Aminobiphenyl (ABP, 5 mg) given i.p. to rat had elimination half-lives of 15.6, 17 and 17 h, respectively, for urinary, faecal and total 3H elimination. 14C-ABP administered orally to rats at 100 mg/kg gave elimination half-lives of 31, 36.7 and 34 h, respectively, for urinary, faecal and total 14C elimination. 2. Semi-log plots of percentage dose remaining in the body versus time indicated that: (i) 82% of 3H activity was excreted in 36 h with a half-life of 14.4 h and 18% with a half-life of 46.2 h, and (ii) 77% of 14C activity was excreted in 48 h with a half-life of 15 h and 23% with a half-life of 180 h. 3. After i.p. injection of 10 mg/kg 14C-ABP to rats, ferrihaemoglobin (HbFe3+) concn increased to 60% in 2 h, accompanied by accumulation of 14C activity in erythrocytes, indicating that the active metabolite, N-hydroxy-4-aminobiphenyl (N-hydroxy-ABP) had oxidized haemoglobin-Fe2+ (HbFe2+) and was bound to the erythrocyte. 4. ABP given i.p. to rats at 0.24 mmol/kg rapidly appeared in blood, disappeared with a half-life of 30 min, and blood concn plateaued at 30 nmol/ml. The concn of 4-acetyl-aminobiphenyl (AABP) plateaued at 17 nmol/ml after 15 min, indicating a dynamic equilibrium between N-acetylation of ABP and N-deacetylation of AABP. The concn of 4'-hydroxy-4-acetylaminobiphenyl (4'-hydroxy-AABP) increased slowly at 1.65 nmol/h. 5. AABP given i.p. to rats at 0.88 mmol/kg slowly appeared in the blood, accompanied by the appearance of ABP and 4'-hydroxy-AABP and formation of HbFe3+. After 4 h the concn of AABP and ABP was 27-35 mmol/ml, indicating a dynamic equilibrium between N-deacetylation of AABP and acetylation of ABP. Neither N-hydroxy-ABP nor N-hydroxy-4-acetylaminobiphenyl (N-hydroxy-AABP) were found.

4-Aminobiphenyl hemoglobin adducts in fetuses exposed to the tobacco smoke carcinogen in utero.

Maternal-fetal exchange of a potent tobacco-related human carcinogen, 4-aminobiphenyl, was studied in smoking (n = 14) and nonsmoking (n = 38) pregnant women. N-Hydroxy-4-aminobiphenyl, the active metabolite of 4-aminobiphenyl, forms chemical addition products (adducts) with hemoglobin. Levels of 4-aminobiphenyl hemoglobin adducts were measured in maternal-fetal paired blood samples obtained from smoking and nonsmoking women during labor and delivery. Carcinogen-hemoglobin adducts were detected in all maternal and fetal blood samples. Levels of such adducts were significantly higher (P less than .001) in maternal and fetal blood samples from smokers: the mean 4-aminobiphenyl hemoglobin adduct level was 92 +/- 54 pg/g of hemoglobin in blood samples from fetuses of smokers, and 17 +/- 13 pg/g of hemoglobin in blood samples from fetuses of nonsmokers; the mean maternal 4-aminobiphenyl hemoglobin adduct level was 183 +/- 108 pg/g of hemoglobin in smokers, and 22 +/- 8 pg/g of hemoglobin in nonsmokers. Fetal carcinogen-adduct levels were consistently lower than maternal levels: the mean maternal to fetal ratio was 2.4 +/- 1.1 in smokers and 1.9 +/- .98 in nonsmokers. Fetal 4-aminobiphenyl hemoglobin adduct levels were strongly associated (correlation coefficient [r2] = .51, P = .002) with maternal 4-aminobiphenyl hemoglobin adduct levels when paired samples from smoking mothers were analyzed. A measure of third-trimester tobacco smoke exposure based on number of cigarettes smoked per day, amount of each cigarette smoked, and depth of inhalation was associated (r2 = .59, P = .029) with maternal 4-aminobiphenyl levels but not with fetal 4-aminobiphenyl levels. This study demonstrates that a potent tobacco-related carcinogen, 4-aminobiphenyl, or its active metabolite, N-hydroxy-4-aminobiphenyl, crosses the human placenta and binds to fetal hemoglobin in concentrations that are significantly higher in smokers than in nonsmokers.

Haemoglobin adducts formed by aromatic amines in smokers: sources of inter-individual variability.

In a previous study we found that aromatic amines, particularly 4-aminobiphenyl, formed haemoglobin adducts at higher concentrations in the blood of smokers compared to non-smokers. We re-analyse here data on haemoglobin adducts of 14 aromatic amines in order to ascertain if the inter-individual variability left unexplained by tobacco smoking could be attributed to differences in individual metabolic patterns. For this purpose we computed residuals from analysis of variance in order to adjust for individual smoking habits (type and amount of tobacco). Residuals were correlated within two clearly distinct groups: one formed by binuclear compounds (4-aminobiphenyl, 3-aminobiphenyl and 2-naphthylamine) and the other formed by all other (i.e. mononuclear) compounds. Within each group, highly statistically significant correlation coefficients were found, whereas compounds belonging to one group were not correlated to compounds in the other group. These results can be interpreted as a suggestion that two different metabolic pathways exist, one for binuclear and one for mononuclear arylamines, and that inter-individual differences in such pathways can explain part of inter-individual variability in adduct levels. This interpretation is consistent with recent animal experiments suggesting that there are different enzyme systems for the two classes of compounds.

Elevated blood levels of carcinogens in passive smokers.

The hypothesis that involuntary exposure to tobacco smoke--passive smoking--results in greater risk of cancer was assessed by measuring the levels of two known carcinogens in the blood of 57 nonsmokers with varying degrees of involuntary exposure, including six heavily exposed bartenders. The concentrations of hemoglobin adducts of 4-aminobiphenyl, a bladder carcinogen, were significantly higher in subjects with confirmed involuntary exposure (plasma cotinine concentrations between 2 and 23 ng/ml) compared with subjects with undetectable levels of cotinine. Similarly, adducts of 3-aminobiphenyl were significantly elevated in subjects with confirmed exposure. The odds of 3-aminobiphenyl adduct levels exceeding 2 pg/g of hemoglobin were 6:7 among the confirmed exposed, compared with the odds of 2:42 among subjects with undetectable cotinine (odds ratio = 18; 95 percent confidence interval = 3.3, 94). The validity of the assay was demonstrated by showing striking declines in adduct levels among quitting smokers.

On the maternal transfer of 4-aminobiphenyl in rats.

The potential for 4-aminobiphenyl (4-ABP) to be transferred from circulating blood into the milk of lactating Sprague-Dawley rats was determined. The distribution of 14C-labeled 4-ABP into milk was examined at time intervals of less than 1, 20, 60, 120, 240 and 480 min after i.v. dose administration. Elimination of radioactivity from blood and milk was determined to be biphasic. The levels of 4-ABP and/or metabolites were lower in milk than in blood at all time points examined. The levels of radioactivity detected in blood declined less rapidly than in milk. That is, the percent of the dose per ml of blood declined from 0.81-0.45, while the percent of the dose per ml of milk declined from 0.38-0.06 during the 8 h time period. The radioactivity present in milk was partially extractable with ethyl acetate with 43% of the radioactivity being extractable at the earliest time point while only 16% was extractable after 8 h. The level of radioactivity associated with the protein precipitate of the milk samples increased from 4-21% within 4 h after treatment. The potential of 4-ABP or its metabolites to exert a genotoxic effect on newborn pups via maternal transfer was also examined. Dams were treated on day 1 post partum and then daily with 4-ABP (10 mg/kg) in corn oil or corn oil alone for 2 weeks. Each experimental group had four liters of pups each containing 5 pups. Pups were sacrificed at 15 days of age, separated by sex and the levels of 4-ABP:DNA adducts in liver determined using 32P-postlabeling. DNA adduct profiles were similar between male and female pups with total adduct levels of 332 and 338 fmol of adducts/mg of DNA, respectively. These results indicate that the genotoxic effects of 4-ABP can be transmitted from exposed dams to the nursing offspring.