Comparison of DNA-adduct and tissue-available dose levels of MeIQx in human and rodent colon following administration of a very low dose

[2-14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was administered orally (304 ng/kg body-weight dose based upon an average 70-kg-body-weight subject) to 5 human colon-cancer patients (58 to 84 years old), as well as to F344 rats and B6C3F1 mice. Colon tissue was collected from the human subjects at surgery and from the rodents 3.5 to 6 hr after administration. Colon DNA-adduct levels and tissue available doses were measured by accelerator mass spectrometry (AMS). The mean levels of MeIQx in the histologically normal colon tissue were not different among the human (97 +/- 26 pg MeIQx/g), rat (133 +/- 15 pg/g) or mouse (78 +/- 10 pg/g) tissues; and no difference existed between the levels detected in human normal and tumor tissue (101 +/- 15 pg/g). Mean DNA-adduct levels in normal human colon (26 +/- 4 adducts/10(12) nucleotides) were significantly greater (p < 0.01) than in rats (17.1 +/- 1 adduct/10(12) nucleotides) or mice (20.6 +/- 0.9 adduct/10(12) nucleotides). No difference existed in adduct levels between normal and tumor tissue in humans. These results show that MeIQx forms DNA adducts in human colon at low dose, and that the human colon may be more sensitive to the effects of MeIQx than that of mice or rats.     

A new assay for albumin and hemoglobin adducts of 1,2- and 1,4-benzoquinones

A new method has been developed to detect mono-S-substituted cysteinyl adducts of 1,2- and 1,4-benzoquinone (BQ) in hemoglobin (Hb) and albumin (Alb). After reacting the protein with trifluoroacetic anhydride and methanesulfonic acid, the resulting isomers of O,O',S-tris-trifluoroacetyl-hydroquinone and -catechol are extracted and detected by gas-chromatography-mass spectrometry in the negative-ion chemical ionization mode. The limit of detection of the assay is about 20 pmol adduct/g protein. This assay was employed to quantitate mono-S-substituted background adducts in human and rat Hb and Alb and benzene-specific adducts in Hb and Alb from F344 rats following a single oral dosage of 50-400 mg [13C6]benzene/kg body wt. In Alb, a dose-related increase in both [13C6]1,2- and [13C6]1,4-BQ adducts was observed with [[13C6]]1,4-BQ-Alb] > [[13C6]1,2-BQ-Alb]. The formation of [13C6]1,2-BQ-Alb was linear with increasing dosage of benzene with a slope of 2.3 (pmol adduct/g protein)/(mg/kg body wt.) (S.E. = 0.18, R2 = 0.91). However, at dosages above about 100 mg [13C6]benzene/kg body wt., the levels of 1,4-BQ-Alb were greater than proportional to the dosage. Mono-S-substituted adducts of [13C6]1,2-BQ and [13C6]1,4-BQ were not detected in Hb. The background ([12C6]) adducts of 1,2- and 1,4-BQ in 20 F344 rats were estimated (in nmol adduct/g of protein) to be 3.9 (S.E. = 0.23) and 4.9 (S.E. = 0.30) in Hb and 2.7 (S.E. = 0.24) and 11.4 (S.E. = 0.60) in Alb. At the highest dosage of 400 mg [13C6]benzene/kg body wt., background levels of 1,2-BQ-Alb were about 4-fold higher than those of the benzene-specific adducts whereas the benzene-specific levels of 1,4-BQ-Alb were about 7-fold higher than those of the background adducts. Background levels of 1,2- and 1,4-BQ adducts in 10 portions of commercial human proteins were found to be (in nmol adduct/g of protein) 1.6 (S.E. = 0.05) and 0.85 (S.E. = 0.04) in Hb and 1.6 (S.E. = 0.06) and 8.9 (S.E. = 0.36) in Alb.     

Disposition of [14C]avitriptan in rats and humans

Avitriptan is a new 5-HT1-like agonist with abortive antimigraine properties. The study was conducted to characterize the pharmacokinetics, absolute bioavailability, and disposition of avitriptan after intravenous (iv) and oral administrations of [14C]avitriptan in rats and oral administration of [14C]avitriptan in humans. The doses used were 20 mg/kg iv and oral in the rat, 10 mg iv in humans, and 50 mg oral in humans. The drug was rapidly absorbed after oral administration, with peak plasma concentrations occurring at 0.5 hr postdose. Absolute bioavailability was 19.3% in rats and 17.2% in humans. Renal excretion was a minor route of elimination in both species, with the majority of the dose being excreted in the feces. After a single oral dose, urinary excretion accounted for 10% of the administered dose in rats and 18% of the administered dose in humans, with the remainder excreted in the feces. Extensive biliary excretion was observed in rats. Avitriptan was extensively metabolized after oral administration, with the unchanged drug accounting for 32% and 22% of the total radioactivity in plasma in rats and humans, respectively. Plasma terminal elimination half-life was approximately 1 hr in rats and approximately 5 hr in humans. The drug was extensively distributed in rat tissues, with a tendency to accumulate in the pigmented tissues of the eye.     

Accumulation of DNA adducts of 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) in tissues and white blood cells of the Fischer-344 rat after multiple oral dosing

The genotoxic effect of an environmental chemical may be estimated from the concentration of its DNA adducts in peripheral white blood cells (WBCs). The food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is carcinogenic in the Fischer-344 rat, affecting principally the liver, small intestine and large intestine. In the present study we have determined whether DNA adducts of IQ are present in circulating WBCs of rats after single or multiple oral doses of IQ and how these adducts are related to those in internal organs. Male Fischer-344 rats received IQ as an oral dose (5 or 50 mg/kg, starting on day 0) by daily gavage (1, 8 or 15 days of treatment). Using 32P-postlabeling assays, IQ-DNA adducts were isolated and quantitated in organs and WBCs on days 1, 8 and 15. Adduct patterns in WBCs were qualitatively similar to those in the organs and adduct formation was highest in the liver, followed by the lungs, kidneys, stomach, large intestine, WBC and small intestine. Accumulation of adducts occurred in all organs and in WBCs in a dose- and time-dependent manner. For all organs, IQ-DNA adduct formation was strongly correlated with those in WBCs. It is concluded that IQ-DNA adducts in WBCs are qualitatively and quantitatively directly related to those in internal organs, independent of the target organ specificity of the carcinogenic effect of IQ.     

The disposition of allyl isothiocyanate in the rat and mouse

The urine was the major route of excretion of radioactivity (50-80% of dose) following the oral administration (2.5 and 25 mg/kg body weight) of allyl[14C]isothiocyanate (AITC) to male and female Fischer 344 rats and B6C3F1 mice. Smaller amounts were found in the faeces (6-12%) and expired air (3-7%). The major difference between the two species was the greater retention of radioactivity after 4 days within rats (18-24% of dose) when compared with mice (2-5% of dose). Three radioactive components were found in the urine of mice and two in rats. The three components were inorganic thiocyanate, allylthiocarbamoylmercapturic acid and allylthiocarbamoylcysteine in mice, but no cysteine conjugate was found in rat urine. In the mouse, approximately 80% of the 14C was present in the urine as the thiocyanate ion whereas in the rat some 75% was as the mercapturate. This indicates that in the mouse, hydrolysis of AITC was the major metabolic pathway whereas in the rat glutathione conjugation was the major route. A species difference was seen in the amount of [14C]AITC-derived radioactivity present in the whole blood of rats and mice; measurable levels of radioactivity remained within rat blood for a longer time period (up to 240 hr) when compared with mice (96 hr). Examination of the urinary bladders of male and female rats following oral dosing with [14C]AITC showed a sex difference with greater amounts of [14C]AITC and/or its metabolites within the bladder tissue of male rats. This data is discussed in terms of the known species- and sex-specificity of the urinary bladder tumours, which occurred after long-term administration to male rats, but not to female rats or mice of either sex, in a carcinogenicity study conducted by the National Toxicology Program in the USA.     

A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and excretion in the urine of NPYR-treated rats

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endogenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of alpha-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. alpha-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 degrees C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.00004% of the dose as adduct 8. The major differences in product formation in reactions of alpha-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.     

Hemoglobin adducts of benzo[a]pyrene diolepoxide in newspaper vendors: association with traffic exhaust

Benzo[a]pyrene diol epoxide adducts with hemoglobin (Hb) were measured to detect human exposure to environmental benzo[a]pyrene from traffic exhaust. Benzo[a]pyrene tetrahydrotetrols (BPTs) released from Hb after acid hydrolysis were quantitated by gas chromatography-mass spectrometry after immunoaffinity chromatography. Fifty three newspaper vendors were enrolled. The median adduct concentration was 0.3 fmol BPTs/mg Hb in high density traffic-exposed vendors and < or = 0.1 fmol BPTs/mg Hb in those exposed to low density traffic; the difference was not significant (P = 0.09). Among non-smokers, adducts were detectable in 60% of high exposure subjects (median 0.3 fmol BPTs/mg Hb) and in 28% of those with low exposure (median < or = 0.1 fmol/mg Hb). This difference was significant (P = 0.02). In low exposure smokers the median of adducts was 0.26 fmol BPTs/mg Hb, while in low exposure non-smokers it was < or = 0.1 fmol BPTs/mg Hb (P = 0.08, not significant). Adduct concentration was no different for low and high density traffic-exposed smokers (P = 0.82). The data indicate a significant difference in adduct concentration related to traffic exhaust exposure among non-smokers.     

Acetylator genotype-dependent formation of 2-aminofluorene-hemoglobin adducts in rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus

Arylamine-hemoglobin adducts are a valuable dosimeter for assessing arylamine exposures and carcinogenic risk. The effects of age, sex, time-course, dose, and acetylator genotype on levels of 2-aminofluorene-hemoglobin adducts were investigated in homozygous rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Pats) acetylator hamsters congenic at the polymorphic (NAT2) acetylator locus. Following administration of a single ip dose of [3H]2-aminofluorene, peak 2-aminofluorene-hemoglobin adduct levels were achieved at 12-18 hr and retained a plateau up to 72 hr postinjection in both rapid and slow acetylator congenic hamsters. 2-Aminofluorene-hemoglobin adduct levels did not differ significantly between young (5-6 weeks) and old (32-49 weeks) hamsters or between male and female hamsters within either acetylator genotype. 2-Aminofluorene-hemoglobin adduct levels increased in a dose-dependent manner (r = 0.95, p = 0.0001) and were consistently higher in slow versus rapid acetylator congenic hamsters in studies of both time-course and dose-effect. The magnitude of the acetylator genotype-dependent difference was a function of dose; 2-aminofluorene-hemoglobin adduct levels were 1.5-fold higher in slow acetylator congenic hamsters following a 60 mg/kg 2-aminofluorene dose (p = 0.0013) but 2-fold higher following a 100 mg/kg 2-aminofluorene dose (p < 0.0001). These results show a specific and significant role for NAT2 acetylator genotype in formation of arylamine-hemoglobin adducts, which may reflect the relationship between acetylator genotype and the incidence of different cancers from arylamine exposures.     

Effects of 16-week dietary administration of 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline in rats

2-Amino-3,8-dimethylimidazo[4, 5-f]quinoxaline (MeIQx), a heterocyclic amine found in cooked meats, is carcinogenic in mice and rats at high doses. In order to examine the toxicity including preneoplastic changes at the lower doses, a total of 170 male Fischer 344 rats were administered MeIQx for 16 weeks at a dose of 100, 10, 1, 0.1, 0.01, 0.001 ppm or 0 ppm in the diet. The numbers of GST-P positive foci and BrdU-labeling indices in the liver were significantly increased by the dietary administration of 10 ppm and 1 ppm or more of MeIQx respectively, when compared with the basal diet-fed control rats. Aberrant cry p tfoci (ACF) were also significantly increased in the 100 ppm MeIQx group as compared to the control value. No histopathological changes indicating obvious toxicity of MeIQx were observed in the major organs other than the liver and large intestine. In conclusion, our results clearly indicate that MeIQx selectively targets the liver and large intestine of rats as organs for the toxicity, but dose not affect the other major organs at low doses.     

Immunochemical identification of hepatic protein adducts derived from estragole

Hepatic protein adducts derived from the allylbenzene food flavor estragole, which is hepatocarcinogenic when given to rodents at high doses, have been identified using immunochemical approaches. Male Fischer 344 rats were given estragole orally and hepatic protein adducts were detected by immunoblotting, using antisera raised by immunizing rabbits with 4-methoxycinnamic acid-modified rabbit serum albumin. A major 155-kDa adduct was expressed in livers of animals that had been treated with estragole at 100, 300, or 500 mg/kg. Levels of expression of the adduct increased disproportionately with respect to dose, and other adducts (170, 100, 44, and 35 kDa) were detected also in the high-dose group. Rats given estragole for 5 days, at 300 mg/kg/day, expressed predominantly 155- and 44-kDa adducts. The 155-, 100-, 44-, and 35-kDa adducts were detected in greatest abundance in liver microsomal fractions, while the 170-kDa adduct was most abundant in the nuclear fraction. Interestingly, whereas the 170-, 155-, 100-, and 35-kDa adducts were detected in cytosolic fractions, relatively low levels of the 44-kDa adduct were detected in nuclear fractions but not in cytosolic fractions. The various adducts were solubilized when microsomal fractions were extracted with sodium carbonate and were digested by trypsin. This implies that the target proteins are peripheral membrane proteins bound to the outer surface of microsomal membranes. Experiments undertaken with isolated rat hepatocytes and with V79 cells transfected with human monoamine phenol sulfotransferase cDNA revealed that adduct formation required 1'-hydroxylation of estragole, followed by sulfation. The pattern of adducts expressed when the transfected V79 cells were incubated with 1'-hydroxyestragole was very similar to that expressed in livers of estragole-treated rats. These cells should constitute a valuable in vitro model system for investigation of toxicological consequences arising from estragole-induced protein adduct formation.     

Effect of glutathione depletion on exocyclic adduct levels in the liver DNA of F344 rats

The effects of glutathione (GSH) depletion on the in vivo formation of cyclic 1,N2- propanodexoxyguanosine adducts (AdG and CdG) as background lesions in the liver DNA of F344 rats were investigated. A group of 5 male F344 rats were given drinking water containing 30 mM L-buthionine (S,R)-sulfoximine (BSO) for 21 days, and another group of 8 rats were given only drinking water as controls. The BSO-treated rats had significantly lower weight gain than control rats. The hepatic GSH levels in the BSO-treated group were reduced by 84% as compared with the control group, from 4.43 to 0.72 mumol/g of tissue. The isomeric AdG3, CdG1, and CdG2 were detected by the 32P-postlabeling/HPLC method in the liver DNA of rats without carcinogen treatment, as we reported previously [Nath, R. G., and Chung, F.-L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495. Nath, R. G., et al. (1996) Cancer Res. 56, 452-456]. The mean levels (mumol/mol of guanine) for AdG3, CdG1, and CdG2 were 0.57 +/- 0.25, 0.15 +/- 0.18, and 0.16 +/- 0.22 for the control group and 1.18 +/- 1.03, 3.16 +/- 3.26, and 2.50 +/- 2.59 for the BSO group, respectively. These increases correspond to approximately 2-fold for AdG and 15-21-fold for CdG adducts. The dramatic increase in the cyclic adduct levels in rat liver DNA could have resulted mainly from GSH depletion as a result of the BSO treatment, even though other unknown effects due to the toxicity of BSO cannot be ruled out. These results suggest that GSH plays an important role in protecting the liver against cyclic propano DNA adduction and provide further support for the endogenous origin of these adducts.     

Developmental regulation of the 3-methylcholanthrene- and dioxin-inducible CYP1A5 gene in chick embryo liver in vivo

Measurement of specific adducts to hemoglobin can be used to establish the dosimetry of electrophilic compounds and metabolites in experimental animals and in humans. The purpose of this study was to investigate the dose response for adduct formation and persistence in rats and mice during long-term low-level exposure to butadiene by inhalation. Adducts of 3,4-epoxy-1-butene, the primary metabolite of butadiene, with N-terminal valine in hemoglobin were determined in male B6C3F1 mice and male Sprague-Dawley rats following exposure to 0, 2, 10, or 100 ppm of 1,3-butadiene, 6 h/day, 5 days/week for 1, 2, 3, or 4 weeks. Blood samples were collected from groups of five mice and three rats at the end of each week during the 4 weeks of exposure and weekly for 3 weeks following the end of the 4-week exposure period. The increase and decrease, respectively, of the adduct levels during and following the end of the 4-week exposure followed closely the theoretical curve for adduct accumulation and removal for rats and mice, thereby demonstrating that the adducts are chemically stable in vivo and that the elimination follows the turnover of the red blood cells. The adduct level increased linearly with butadiene exposure concentration in the mice, whereas a deviation from linearity was observed in the rats. For example, after exposure to 100 ppm butadiene, the epoxybutene-hemoglobin adduct levels were about four times higher in mice than in rats; at lower concentrations of butadiene, the species difference was less pronounced. Blood concentrations of epoxybutene, estimated from hemoglobin adduct levels, were in general agreement with reported concentrations in mice and rats exposed by inhalation to 62.5 ppm. These studies show that adducts of epoxybutene with N-terminal valine in hemoglobin can be used to predict blood concentration of epoxybutene in experimental animals.     

Benzo[b]fluoranthene: tumorigenicity in strain A/J mouse lungs, DNA adducts and mutations in the Ki-ras oncogene

The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F) is a pervasive constituent of environmental combustion products. We sought to examine the lung tumorigenic activity of B[b]F in strain A/J mice, to study the relationship between formation and decay of B[b]F-DNA adducts and to examine mutations in the Ki-ras proto-oncogene in DNA from B[b]F-induced tumors. Mice were given i.p. injections of 0, 10, 50, 100 or 200 mg/kg body wt and lung adenomas were scored after 8 months. B[b]F induced significant numbers of mouse lung adenomas in a dose-related fashion, with the highest dose (200 mg/kg) yielding 6.95 adenomas/ mouse, with 100% of the mice exhibiting an adenoma. In mice given tricaprylin, the vehicle control, there were 0.60 adenomas/mouse, with 55% of the mice exhibiting an adenoma. Based on dose, B[b]F was less active than benzo[a]pyrene. DNA adducts were analyzed qualitatively and quantitatively by 32P-post-labeling in lungs of strain A/J mice 1, 3, 5, 7, 14 and 21 days after i.p. injection. Maximal levels of adduction occurred 5 days after treatment with the 200 mg/kg dose group, producing 1230 amol B[b]F-DNA adducts/microgram DNA. The major B[b]F-DNA adduct was identified by co-chromatography as trans-9, 10-dihydroxy-anti-11, 12-epoxy-5-hydroxy-9, 10, 11, 12-tetra-hydro-B[b]F-deoxyguanosine. Approximately 86% of the tumors had a mutation in codon 12 of the Ki-ras oncogene, as determined by direct DNA sequencing of PCR-amplified exon 1 and single-stranded conformation polymorphism analysis. Analysis of the Ki-ras mutation spectrum in 25 of 29 B[b]F-induced tumors revealed the predominant mutation to be a G-->T transversion in the first or second base of codon 12, congruous with the DNA adduct data. Our data are consistent with previous reports in mouse skin implicating a phenolic diol epoxide as the proximate carcinogenic form of B[b]F that binds to guanine.     

The association of non-insulin-dependent diabetes mellitus and cognitive function in an older cohort

This is the first demonstration of the use of accelerator mass spectrometry (AMS) as a tool for the measurement of 3H with attomole (10(-18) mol) sensitivity in a biological study. AMS is an analytical technique for quantifying rare isotopes with high sensitivity and precision and has been most commonly used to measure 14C in both the geosciences and more recently in biomedical research. AMS measurement of serially diluted samples containing a 3H-labeled tracer showed a strong correlation with liquid scintillation counting. The mean coefficient of variation of 3H AMS based upon the analysis of separately prepared aliquots of these samples was 12%. The sensitivity for 3H detection in tissue, protein, and DNA was approximately 2-4 amol/mg of sample. This high sensitivity is comparable to detection limits for 14C-labeled carcinogens using 14C AMS and demonstrates the feasibility of 3H AMS for biomedical studies. One application of this technique is in low-dose, dual-isotope studies in conjunction with 14C AMS. We measured the levels of 3H-labeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 14C-labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rat liver tissue and bound to liver DNA and protein 4.5 h following acute administration of individual or coadministered doses in the range of 4-5100 pmol/kg of body weight. Levels of PhIP and MeIQx in whole tissue and bound to liver protein were dose-dependent. MeIQx-protein and -DNA adduct levels were higher than PhIP adduct levels, which is consistent with their respective carcinogenicity in this organ. Coadministration of PhIP and MeIQx did not demonstrate any measurable synergistic effects compared to administration of these compounds individually. These studies demonstrate the application of AMS for the low-level detection of 3H in small biological samples and for its use in conjunction with 14C AMS for dual-labeling studies.     

DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and a neurotoxic metabolite of o-tolyl phosphates. In a previous paper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is mutagenic in Salmonella typhimurium TA100 and forms DNA adducts in incubations with nucleotides, nucleosides and isolated DNA. In the present study we compare DNA adduct formation using 32P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide-treated bacteria (S.typhimurium TA100) and hepatoma cells with DNA adducts formed in liver, kidney, lung and heart of tri-o-tolyl phosphate-exposed Fischer 344 male rats. In both bacteria and hepatoma cells two DNA adducts could be detected after treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide. The minor adduct co-chromatographed with synthetic N3-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling. The major DNA adduct was a cytidine adduct, most likely N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate. Male Fischer 344 rats were treated orally for 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNA was isolated from liver, kidney, lung, heart, brain and testes 1, 4, 7 and 28 days after giving the last dose. Analysis by 32P-postlabelling revealed that two adducts were present in the DNA isolated from liver, kidney, lung and heart on the first day after giving the last dose; DNA adducts were not detected in the brain and testes. The adduct pattern after in vivo treatment with tri-o-tolyl phosphate was identical with that found in bacteria and hepatoma cells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin 2-oxide, the major adduct being N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate and the minor N3-(o-hydroxybenzyl)deoxyuridine 3' monophosphate. Both DNA adducts persisted in the lungs for the entire observation period, whereas in the kidney only the cytidine adduct could be detected 28 days after the last dose of tri-o-tolyl phosphate. In liver and heart the adducts were detectable only on the first day after completion of the treatment. The results indicate that in addition to the well established neurotoxicity, some o-tolyl phosphates may have a carcinogenic potential.     

Genotoxicity of coke-oven and urban air particulate matter in in vitro acellular assays coupled with 32P-postlabeling and HPLC analysis of DNA adducts

This study is an in vitro part of the ongoing biomarker studies with population from a polluted region of Northern Bohemia and coke-oven workers from Czech and Slovak Republics. The aim of this study is to compare DNA adduct forming ability of chemical compound classes from both the urban and coke-oven extractable organic mass (EOM) of airborne particles. The crude extracts were fractionated into seven fractions by acid-base partitioning and silica gel column chromatography. In in vitro acellular assays we used calf thymus DNA (CT DNA) with oxidative (+S9) and reductive activation mediated by xanthine oxidase (+XO) under anaerobic conditions. Both the butanol and nuclease P1 versions of 32P-postlabeling for detection of bulky aromatic and/or hydrophobic adducts were used. The results showed that the spectra of major DNA adducts resulting from both the in vitro assays are within the fractions similar for both the urban and coke-oven samples. The highest DNA adduct levels with S9-activation were detected for the neutral aromatic fraction, followed by slightly polar and acidic fractions for both samples. With XO-mediated metabolism, the highest DNA adduct levels were detected for both the acidic fractions. Assuming additivity of compound activities, then the acidic fraction, which in the urban sample comprises a major portion of EOM mass (28%), may contain the greatest activity in both in vitro assays (39 and 69%, +S9 and +XO, respectively). In contrast, the aromatic fraction constituting only 8% of total urban EOM mass may account for comparable activity (34%) with organic acids. The highest DNA adduct forming activity of the coke-oven sample accounts for the aromatic fraction (82 and 63%, +S9 and +XO, respectively) that also contains the greatest portion of the total EOM (48%). To characterize some of the specific DNA adducts formed, we coupled TLC on 20x20 cm plates with HPLC analysis of 32P-postlabeled adducts. In both S9-treated samples of the aromatic fraction, we tentatively identified DNA adducts presumably diolepoxide-derived from: 9-hydroxy-benzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide[+/-] (anti-BPDE), benzo[b,j,k]fluoranthenes (B[b]F, B[j]F, B[k]F), chrysene (CHRY), benz[a]-anthracene (B[a]A) and indeno[cd]pyrene (I[cd]P). These DNA adducts accounted for about 57% of total DNA adducts detected in both S9-treated samples of the aromatic fraction. DNA adducts of XO-treated samples were sensitive to nuclease P1 and HPLC profiles of the major adducts were markedly different from the major adducts of S9-treated samples. However, the combination of TLC and HPLC did not confirm the presence of DNA adducts derived from 1-nitropyrene (1 NP), 9-nitroanthracene (9 NA) and 3-nitrofluoranthene (3 NF) that were detected by GC-MS in the slightly polar fraction. We concluded that the chemical fractionation procedure facilitates the assessing of DNA adduct forming ability of different chemical compound classes. However, based on the results obtained with the whole extracts, it does not fulfil a task of the actual contribution of individual fractions within the activity of the whole extracts. Our results are the first in detecting of DNA adducts derived from urban air and coke-oven particulate matter.     

Growth and cuticular synthesis in Ascaris suum larvae during development from third to fourth stage in vitro

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the adduct level ranged from 6.81 to 108.50 adducts/10(8) nucleotide. Thirty-three of 39 (85%) had detectable levels of BPDE-DNA adducts (> 1 adduct/10(9) nucleotide). Current heavy smokers (> 20 cigarettes/day) have significantly higher DRZ adduct levels compared to individuals smoking less than 20 cigarettes/day. By SFS combined with immunoaffinity column (IAC), 11 of 39 (28%) samples had detectable adduct levels, and 6 of 11 (55%) were detectable by SFS following purification of benzo[a]pyrene (BP)-tetrols by high pressure liquid chromatography (HPLC). Six of 33 (18%) samples were positive for BPDE-DNA adducts by both postlabeling and HPLC/SFS. No correlation was observed between the SFS and 32P-postlabeling assays for the detection of BPDE-DNA adducts. However, there was a good correlation between adduct levels detected by IAC/SFS and HPLC/SFS. We found a weak association between total PAH-DNA adduct levels in lung tissue and TP53 mutations.     

Trace elements in human scalp hair and soil in Irian Jaya

Benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) which are present in cigarette smoke, are common air and food genotoxic contaminants and possible human carcinogens. We measured the following PAH levels: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, BaP, dibenzo[a,h]anthracene, benzo[g,h,i]perylene as well as (+/-) syn and anti BaP diol-epoxide (BPDE) DNA adducts in autopsy samples from the lungs of non-smokers, ex-smokers and smokers who had lived in Florence, Italy. PAH levels in lung tissue were similar in all groups, with the exception of dibenzo[a,h]anthracene (DBA), which was higher in lung samples from smokers (n = 10, 0.18+/-0.17 ng/g d.w, mean +/- S.D.) compared to non-smokers (n = 15, 0.046+/-0.025 ng/g d.w) (P < 0.05), whereas ex-smokers (n = 5), had intermediate levels (0.07+/-0.03 ng/g d.w). The average level of total BPDE-DNA adducts was 4.46+/-5.76 per 10(8) bases in smokers, 4.04+/-2.37 per 10(8) in ex-smokers and 1.76+/-1.69 per 10(8) in non-smokers. The levels of non-smokers were significantly different (P < 0.05) from the levels of the smokers and ex-smokers combined. Total BPDE-DNA adducts were correlated with BaP levels in the lung samples in which both determinations were obtained (r = 0.63). Our results demonstrate that the biological load of PAHs due to environmental pollution is similar in individuals who smoke and those who do not, but BPDE-DNA adducts are higher in smokers and ex-smokers compared to non-smokers. This study further confirms the usefulness of BPDE-DNA adduct levels determination in the lungs from autopsy samples for monitoring long-term human exposure to BaP, a representative PAH.     

Absorption, disposition kinetics, and metabolic pathways of cyclohexene oxide in the male Fischer 344 rat and female B6C3F1 mouse

Cyclohexene oxide (CHO) is a monomer intermediate used in the synthesis of pesticides, pharmaceuticals, and perfumes. Although CHO has a variety of industrial uses where direct human exposure is possible, very little is known about its fate in the body. Therefore, the objectives of this study were to determine the absorption, distribution, metabolism, and excretion of cyclohexene oxide after oral, intravenous, and dermal exposure in male Fischer 344 rats and female B6C3F, mice. After intravenous administration of [14C]CHO (50 mg/kg), CHO was rapidly distributed, metabolized, and excreted into the urine. Plasma concentrations of CHO rapidly declined and were below the limit of detection within 60 min. Average (+/- SD) values for terminal disposition half-life, apparent volume of distribution at steady-state, and systemic body clearance were: 19.3 +/- 1.6 min; 0.44 +/- 0.08 liter/kg; and 31.3 +/- 0.5 ml/kg * min, respectively. After oral administration of [14C]CHO (10 and 100 mg/kg), it was found that 14C-equivalents were rapidly excreted in the urine of both species. At 48 hr, the majority of the dose (73-93%) was recovered in urine, whereas fecal elimination accounted for only 2-5% of the dose. At no time after oral administration was parent CHO detected in the blood. However, its primary metabolite cyclohexane-1,2-diol was present for different lengths of time depending on the dose. Four metabolites were detected and identified in mouse urine by MS: cyclohexane-1,2-diol; cyclohexane-1,2-diol-O-glucuronide; N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine; and cyclohexane-1,2-diol-O-sulfate. The sulfate conjugate was not present in rat urine. Topical application of [14C]CHO (60 mg/kg) provided poor absorption in both species. The majority of 14C-equivalents applied dermally were recovered from the charcoal skin trap (approximately 90% of the dose). Only 4% of the dose was absorbed, and the major route of elimination was via the urine. To evaluate the toxicity of CHO, animals were given daily doses of CHO orally and topically for 28 days. No statistically significant changes in final body weights or relative organ weights were noted in rats or mice treated orally with CHO up to 100 mg/kg or up to 60 mg/kg when given topically. Very few lesions were found at necropsy, and none were considered compound related. In conclusion, regardless of route, CHO is rapidly eliminated and excreted into the urine. Furthermore, after either oral or dermal administration, it is unlikely that CHO reaches the systemic circulation intact due to its rapid metabolism, and is therefore unable to cause toxicity in the whole animal under the test conditions used in this study.     

Hemoglobin and albumin adducts of benzene oxide among workers exposed to high levels of benzene

Benzene oxide (BO) reacts with cysteinyl residues in hemoglobin (Hb) and albumin (Alb) to form protein adducts (BO-Hb and BO-Alb), which are presumed to be specific biomarkers of exposure to benzene. We analyzed BO-Hb in 43 exposed workers and 42 unexposed controls, and BO-Alb in a subsample consisting of 19 workers and 19 controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. The adducts were analyzed by gas chromatography-mass spectrometry following reaction of the protein with trifluoroacetic anhydride and methanesulfonic acid. When subjects were divided into controls (n = 42) and workers exposed to < or =31 (n = 21) and >31 p.p.m. (n = 22) benzene, median BO-Hb levels were 32.0, 46.7 and 129 pmol/g globin, respectively (correlation with exposure: Spearman r = 0.67, P < 0.0001). To our knowledge, these results represent the first observation in humans that BO-Hb levels are significantly correlated with benzene exposure. Median BO-Alb levels in these 3 groups were 103 (n = 19), 351 (n = 7) and 2010 (n = 12) pmol/g Alb, respectively, also reflecting a significant correlation with exposure (Spearman r = 0.90, P < 0.0001). The blood dose of BO predicted from both Hb and Alb adducts was very similar. These results clearly affirm the use of both Hb and Alb adducts of BO as biomarkers of exposure to high levels of benzene. As part of our investigation of the background levels of BO-Hb and BO-Alb found in unexposed persons, we analyzed recombinant human Hb and Alb for BO adducts. Significant levels of both BO-Hb (19.7 pmol/g) and BO-Alb (41.9 pmol/g) were detected, suggesting that portions of the observed background adducts reflect an artifact of the assay, while other portions are indicative of either unknown exposures or endogenous production of adducts.