Mutagenic activation of aromatic amines by molluscs as a biomarker of marine pollution

Prior studies of the association between socioeconomic status and length of survival among persons infected with the human immunodeficiency virus (HIV) have produced conflicting results. To investigate this issue further, the authors examined data on 18,167 San Francisco, California, residents aged 13 years or older who were diagnosed with acquired immunodeficiency syndrome (AIDS) between January 1, 1985, and December 31, 1995. Three validated US census-based measures of socioeconomic status were used: poverty, predominantly working class neighborhood, and low educational level. Median length of survival was found to be similar for persons living in neighborhoods characterized by poverty (22 months) and those in higher income neighborhoods (23 months), for persons living in predominantly working class neighborhoods (22 months) and those in predominantly professional/managerial neighborhoods (23 months), and for persons living in neighborhoods characterized by low educational level (23 months) and those in neighborhoods characterized by higher educational level (23 months). After adjustment for sex, age, ethnicity, AIDS risk group, site of AIDS diagnosis, time period of AIDS diagnosis, and AIDS-indicator illness, no association was found between survival and living in a neighborhood characterized by poverty (relative hazard (RH)=1.03, 95% confidence interval (CI) 0.97-1.08), between survival and working class occupations (RH=1.03, 95% CI 0.98-1.08), or between survival and low educational level (RH=0.96, 95% CI 0.90-1.01). The lack of an association between socioeconomic status and length of survival with AIDS may be due to the high mortality from AIDS in the era prior to highly effective antiretroviral therapy or to similar levels of access to care in San Francisco.     

Hemoglobin binding of bicyclic aromatic amines

Aromatic diamino compounds, e.g., 4,4'-methylenebis(2-chloroaniline) (MOCA) and 4,4'-methylenedianiline (MDA), are used as curing agents in the production of elastomers. Since MOCA and MDA are mutagenic and carcinogenic, substitutes are of great commercial interest. For benzidine it has been shown that ortho substitution with methyl groups yields the nonmutagenic 3,3',5,5'-tetramethylbenzidine. Therefore, MDA analogues with large substituents in the ortho position have been synthesized. The substituents are supposed to inhibit the formation of the N-hydroxyarylamines which are the putative genotoxic intermediates. We investigated the biological availability of the N-hydroxylamines of ortho-substituted diamines and of known carcinogenic diamines in female Wistar rats, by determining hemoglobin (Hb) adducts. Hb from rats dosed with 0.5 mmol/kg diamine and from controls was isolated and hydrolyzed in base. The released diamine and monoacetyldiamine were quantified by HPLC with electrochemical detection and/or GC/MS. MDA, 4,4'-oxydianiline (ODA), 4,4'-ethylenedianiline, and 4,4'-thiodianiline (TDA) bound to hemoglobin as diamine and as monoacetyl-diamine. 4,4'-Methylenebis(2,6-dimethylaniline), 4,4'-methylenebis(2,6-diethylaniline), MOCA, and 4,4'-sulfonyldianiline (dapsone) bound only as diamine to Hb. 4,4'-Methylenebis(2,6-dichloroaniline) did not bind to Hb. Thus, the presence of two substituents in the ortho position and the presence of electron-withdrawing groups in the para position to the amino group drastically reduced the formation of Hb adducts. The amount of hemoglobin adducts was compared to their carcinogenic potency. The extent of hemoglobin binding of the bicyclic diamines (dapsone, 3,3'-dichlorobenzidine, MDA, MOCA, TDA, ODA, and benzidine) increases with their carcinogenic potency.     

Metabolic activation and carcinogen-DNA adduct detection in human larynx

Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.     

Primary aromatic amines: their N-oxidative bioactivation

There exists a diversity of pathways in mammalian cells serving to activate primary aromatic amines. 1 N-Oxidative mixed-function turnover usually involves participation of the cytochrome P450 superfamily, while catalysis by the flavin-containing monooxygenases is restricted to a few amines capable of forming imine tautomers. Surprisingly, haemoglobin metabolizes cytotoxic and carcinogenic arylamines via a monooxygenase-like mechanism, but peroxygenase activity is also operative. 2 In extrahepatic tissues that exhibit only a low level of monooxygenases, peroxidative transformations, as are brought about by prostaglandin H synthase, myeloperoxidase or lactoperoxidase, predominate in amine activation. Non-mammalian peroxidases frequently used as model systems include horseradish peroxidase and chloroperoxidase. 3 Non-enzymatic, light-induced conversion of aromatic amines to free radical or N-oxy products proceeds either via direct photolysis of the nitrogenous compounds or through attack by lipid-derived reactive intermediates generated during irradiation. 4 The interplay of the various tissue-specific processes of arylamine activation serves to explain differences in susceptibility toward the biological actions of primary aromatic amines.     

Substituent effect on the oxidation of phenols and aromatic amines by horseradish peroxidase compound I

A stopped-flow kinetic study shows that the reduction rate of horseradish peroxidase compound I by phenols and aromatic amines is greatly dependent upon the substituent effect on the benzene ring. Morever it has been possible to relate the reduction rate constants of monosubstituted substrates by a linear free-energy relationship (Hammett equation). The correlation of log (rate constants) with sigma values (Hammett equation) and the absence of correlation with sigma+ values (Okamoto-Brown equation) can be explained by a mechanism of aromatic substrate oxidations, in which the substrate gives an electron to the enzyme compound I and simultaneously loses a proton. The analogy which has been made with oxidation potentials of phenols or anilines strengthens the view that the reaction is only dependent on the relative ease of oxidation of the substrate. The rate constant obtained for p-aminophenol indicates that a value of 2.3 X 10(8) M-1 S-1 probably approaches the diffusion-controlled limit for a bimolecular reaction involving compound I and an aromatic substrate.     

Nucleic acids-protein interactions. Conformational changes induced by the binding of aromatic amines to polyadenylic acid

The binding of Tryptamine, Serotonine, Phenylethylamine and Histamine to poly(A) in its single stranded form at pH 7 leads to a decrease of its circular dichroism (C.D) amplitude without any appreciable alteration of the shape of the C.D. spectrum. The magnitude of the effect depends on the size of the aromatic ring and decreases in the order : tryptamine greater than tyramine greater than phenylethylamine greather than histamine. A method is described which allows the calculation of association constants from C.D. data. The C.D. amplitude decreases linearly with concentration of bound molecules. Binding of aromatic amines to poly(A) leads to a change in the proton chemical shifts of both the amine and the poly(A) protons. Quantitative analysis of P.M.R. data demonstrates that the shifts of poly(A) protons are linearly related to the concentration of bound molecules.     

Metabolic activation of 2,6-dichlorobenzonitrile, an olfactory-specific toxicant, by rat, rabbit, and human cytochromes P450

The authors posit that cellular edema is the major contributor to brain swelling in diffuse head injury and that the contribution of vasogenic edema may be overemphasized. The objective of this study was to determine the early time course of blood-brain barrier (BBB) changes in diffuse closed head injury and to what extent barrier permeability is affected by the secondary insults of hypoxia and hypotension. The BBB disruption was quantified and visualized using T1-weighted magnetic resonance (MR) imaging following intravenous administration of the MR contrast agent gadolinium-diethylenetriamine pentaacetic acid. To avoid the effect of blood volume changes, the maximum signal intensity (SI) enhancement was used to calculate the difference in BBB disruption. A new impact-acceleration model was used to induce closed head injury. Forty-five adult Sprague-Dawley rats were separated into four groups: Group I, sham operated (four animals), Group II, hypoxia and hypotension (four animals), Group III, trauma only (23 animals), and Group IV, trauma coupled with hypoxia and hypotension (14 animals). After trauma was induced, a 30-minute insult of hypoxia (PaO2 40 mm Hg) and hypotension (mean arterial blood pressure 30 mm Hg) was imposed, after which the animals were resuscitated. In the trauma-induced animals, the SI increased dramatically immediately after impact. By 15 minutes permeability decreased exponentially and by 30 minutes it was equal to that of control animals. When trauma was coupled with secondary insult, the SI enhancement was lower after the trauma, consistent with reduced blood pressure and blood flow. However, the SI increased dramatically on reperfusion and was equal to that of control by 60 minutes after the combined insult. In conclusion, the authors suggest that closed head injury is associated with a rapid and transient BBB opening that begins at the time of the trauma and lasts no more than 30 minutes. It has also been shown that addition of posttraumatic secondary insult-hypoxia and hypotension-prolongs the time of BBB breakdown after closed head injury. The authors further conclude that MR imaging is an excellent technique to follow (time resolution 1-1.5 minutes) the evolution of trauma-induced BBB damage noninvasively from as early as a few minutes up to hours or even longer after the trauma occurs.     

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes

The food-borne carcinogenic and mutagenic heterocyclic aromatic amines undergo bioactivation to the corresponding N-hydroxy (OH)-arylamines and the subsequent N-glucuronidation of these metabolites is regarded as an important detoxification reaction. In this study, the rates of glucuronidation for the N-OH derivatives of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by liver microsomal glucuronosyltransferase were compared to that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl (N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-amino-biphenyl (N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronic acid (UDPGA)-dependent glucuroidation of N-OH-IQ, N-OH-PhIP, N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%, respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg). Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidation of N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20% and 10%, respectively of that measured for N-OH-DMABP (11.2 nmol/min/mg); activity towards N-OH-MeIQx was not detected. Two glucuronide(s) of N-OH-PhIP, designated I and II, were separated by HPLC. Conjugate II was found to be chromatographically and spectrally identical with a previously reported major biliary metabolite of PhIP in the rat, while conjugate I was identical with a major urinary metabolite of PhIP in the dog. Hepatic microsomes from rat, dog and human were found to catalyze the formation of both conjugates. The rat preferentially formed conjugate II (I to II ratio of 1:15), while the dog and human formed higher relative amounts of conjugate I (I to II ratio of 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardment mass spectrometry of conjugates I and II gave the corresponding molecular ions and showed nearly identical primary spectra. However, collision-induced spectra were distinct and were consistent with the identity of conjugates I and II as structural isomers. Moreover, the UV spectrum of conjugate I exhibited a lambda max at 317 nm and was essentially identical to that of N-OH-PhIP, while conjugate II was markedly different with a lambda max of 331 nm. Both conjugates were stable in 0.1 N HCl and were resistant to hydrolysis by rat, dog and human liver microsomal beta-glucuronidases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Escherichia coli lacZ strains engineered for detection of frameshift mutations induced by aromatic amines and nitroaromatic compounds

Escherichia coli lacZ strains CC107-CC111, which detect specific frameshift mutations, were used to study the mutational specificities of 2-nitro-3-methylimidazo[4,5-f] quinoline (NO2-IQ) and rat hepatic S9-activated 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). New constructs were made in which UvrABC-dependent excision repair was eliminated (strains DJ3107-DJ3111), followed by introduction of plasmid pYG219 conferring acetyl CoA:arylamine N-acetyltransferase/acetyl CoA:arylhydroxylamine O-acetyltransferase (NAT/OAT) activity (strains DJ3207-DJ3211). Sensitivity to mutagens was greatly enhanced. The mutational specificity of NO2-IQ was identical to that of the corresponding amine, IQ. The most prominent mutations caused by the two compounds were -2(CGGC) and 1(CG) frameshifts. +1(AT) Frameshifts play a minor role in the pattern of mutational specificity. Induction of all three mutations was similarly influenced by NAT/OAT activation and UvrABC-dependent excision repair. These new tester strains provide an effective tool for the study of aromatic amine mutational specificity and the influences of excision repair and NAT/OAT activation.     

Primary aromatic amines from side-stream cigarette smoke are common contaminants of indoor air

A very sensitive mass-spectrometry method has been developed for the analysis of aromatic amines in tobacco smoke and in indoor air. Cigarettes were smoked with a smoking machine; the amines from the smoke were trapped in a 5% HCl water solution containing internal standards and detected by gas chromatography/mass spectrometry in the selected-ion-monitoring (SIM) mode. The amines measured were the following: aniline, 2-toluidine, 3-toluidine, 4-toluidine, 2-ethylaniline, 3-ethylaniline, 4-ethylaniline, 2,3-dimethylaniline, 2,4-dimethylaniline, 2,5-dimethylaniline, 2,6-dimethylaniline, 1-naphthylamine, 2-naphthylamine, 2-methyl-1-naphthylamine, 2-aminobiphenyl, 3-aminobiphenyl and 4-aminobiphenyl. We analyzed nine brands of cigarettes sold commercially in Italy (Gauloise, Nazionali, Marlboro, Camel, MS, MS mild and MS lights), with and without filter. Main-stream smoke contained a lower amount of aromatic amines than side-stream smoke: the total level of these amines in main-stream smoke ranged from 200 to 1300 ng/cigarette, whereas the level of aromatic amines in side-stream smoke varied from 20,000 to 30,000 ng/cigarette. The smoke of black-tobacco cigarettes had higher levels of aromatic amines compared to light-tobacco cigarettes and the filters significantly reduced aromatic amines in main-stream smoke. We also determined the levels of aromatic amines in ambient air, offices and houses. Some aromatic amines (aniline and toluidine) were detected in ambient air, as well as in rooms of non-smokers. Most measurements showed a considerable contamination of aromatic amines derived from side-stream smoke, which was detected also in parts of the buildings in which smoking was not allowed.     

Metabolic interaction of secondary amines and tertiary amino propiophenones with monoamine oxidase systems

The metabolic interaction of three secondary amines and three nitrogen-containing metabolites of safrole (tertiary amino propiophenones) with rat liver mitochondrial monoamine oxidase systems was studied in vitro employing [7-14C] benzylamine--HCl as a substrate. The two cyclic secondary amines, piperidine and pyrrolidine, showed hyperbolic competitive inhibition while pure competitive inhibition was observed in case of dimethylamine--HCl and three safrole metabolites. Inhibition characteristics for rat liver, kidney and brain mitochondrial monoamine oxidase with two cyclic secondary amines and tertiary amino with two cyclic secondary amines and tertiary amino propiophenones of safrole and elemicin were investigated manometrically using tyramine and serotonin as the substrates.     

Adrenaline in the human pancreas

The present study describes the results of examinations of the noradrenaline and adrenaline concentrations in the human pancreas as well as in a number of other organs. Tissue specimens were obtained at post-mortem examination. Adrenaline was present in small amounts in the cardiovascular system, the liver and the spleen in comparison with the noradrenaline concentration. The pancreas, especially the body of the pancreas, contained, however, considerable amounts of adrenaline. The average adrenaline concentration was approximately 20 times higher in the pancreas than in the other organs examined. The greatest concentration of adrenaline was found in the posterior and superior parts of the body of the pancreas. There was no relationship between the cause of death in the human subjects and the adrenaline concentration in the pancreas and large amounts of adrenaline were also found in tissue specimens of pancreas obtained from long-term diabetic patients. Adrenaline was present in the pancreas of the rat, dog and rabbit but in small amounts in comparison with the noradrenaline concentration.     

Underlying and multiple cause mortality in a cohort of workers exposed to aromatic amines

PURPOSE: To investigate the association of a quality of life-visual function questionnaire with an objective clinical test of visual function. METHODS: We have developed a questionnaire to assess self-reported visual satisfaction in ophthalmic patients suffering from chronic eye conditions causing visual impairment. The questionnaire was administered to 120 patients suffering from age-related cataract, chronic open angle glaucoma, age-related macular degeneration, branch retinal vein occlusion, and presbyopia or minor refractive defects. All the participants also underwent determination of visual acuity, contrast sensitivity, glare, and visual field. RESULTS: The questionnaire has a good reproducibility, a high internal consistency, and is able to discriminate between the different groups of patients. The total questionnaire score is significantly associated with the results of all visual function tests with the exception of glare. When entered into a multiple linear regression model, near visual acuity and contrast sensitivity are still considerably associated with the total questionnaire score. The psychological attitude of the patient towards his/her health problem is also associated with the total average score. CONCLUSIONS: Overall, the model explains 49% of the variance in the average questionnaire score.     

The effect of aromatic and aliphatic amines on copper electrowinning from acidic sulphate electrolyte

Copper electrodeposition in the presence of various types of aromatic and aliphatic amines was studied. The effects of operating variables including organic additives concentrations and temperature on the limiting current were investigated by the potentio-dynamic polarization technique. The effects of amines on the surface tensions of the solutions were measured; the results showed that amines reduce solution surface tension. The adsorption of all inhibitors on copper cathode was found to obey Temkin, Flory-Huggin and kinetic adsorption isotherm. The calculated free energy of adsorption (ΔG_ads.) of inhibitor on copper surface indicated that the adsorption reactions were spontaneous (ΔG_ads. < 0). The thermodynamic activation parameters (E_a, ΔH*, ΔS* and ΔG*) were estimated and discussed. It was found that activation energy values for copper electrodeposition in the inhibited solutions were higher than that for the uninhibited solution. The high inhibitor efficiency was discussed in terms of the strong adsorption of inhibitor molecules on the copper surface.     

Bioelectrochemical monitoring of phenols and aromatic amines in flow injection using novel plant peroxidases

An amperometric flow system combined with a glucose oxidase-mutarotase reactor was optimized and used to determine aromatic amines and phenols using peroxidase-modified graphite electrodes. An increase in currents upon injection of the analyzed substrate was shown to be approximated by a Michaelis-Menten type dependence. The detection limit was calculated as 3 times the noise, and the sensitivity was calculated as Imax/K(m)app. Commercially available horseradish peroxidase was compared with tobacco anionic and peanut cationic peroxidases for determination of aromatic amines and phenols. Detection limits of 10 nM for determination of o-aminophenol and o- and p-phenylenediamine achieved with a tobacco peroxidase-modified electrode give a promise for further improvements in sensitivities and detection limits of biosensors.     

In vitro study of the percutaneous absorption of four aromatic amines using hairless rat skin

Using hairless rat skin maintained in a Franz diffusion cell, the percutaneous penetration of four aromatic amines: para-chloroaniline (PCPA), meta-trifluoromethylaniline (mTFMA), dichloro-3,4-aniline (3,4-DCA) and dichloro-3,5-aniline (3,5-DCA) were studied. The purpose of the studies was to determine the permeation parameters (rate of permeation, permeability rat constant) in order to compare the rate of absorption of the four amines. The results show that the four amines penetrate significantly across the skin, but with different rates. 10 h after in vitro application (2 mg/cm2), the extent of permeation was PCPA > mTFMA > 3,4-DCA > 3,5-DCA.     

Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells

The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed when meat is cooked and are rodent mammary carcinogens. They are thought to be metabolically activated by N-hydroxylation, catalysed by cytochrome P450 (CYP), followed by O-acetylation catalysed by N-acetyltransferases. Primary cultures of human mammary epithelial cells (HMECs) prepared from up to 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP (500 microM) or with N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels of adduct formation in their DNA analysed by 32P-post-labelling. In order to investigate whether pharmacogenetic polymorphisms influence DNA adduct formation, the NAT2 genotype of each individual was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that distinguishes between the wild-type and four variant alleles. Presence of two variant alleles designates a slow NAT2 acetylator, whereas individuals with one or two wild-type alleles are designated fast NAT2 acetylators. Interindividual variations in total DNA adduct levels ranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80), for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96), for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean 9.24). The higher adduct levels observed in cells treated with the N-OH metabolites suggests that N-hydroxylation is the rate-limiting step in HMECs and this may be due to low CYP levels. In contrast, the Phase II reaction catalysed by N-acetyltransferases is probably the major step in the metabolic activation of heterocyclic amines that occurs in the breast. Higher mean levels of heterocyclic amine-DNA adduct formation were detected in the cells of NAT2 fast acetylators compared with slow acetylators, with mean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74 and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74, respectively, following MeIQ treatment, of 7.90 and 5.08, respectively and following N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However, due to the large variations in adduct levels, these differences in mean values were not statistically significant with the limited number of individuals studied. This appears to be the first pilot study to demonstrate interindividual variations in the metabolic activation of heterocyclic amines and their metabolic intermediates in primary cultures of HMECs in vitro.