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Metabolism experiments were conducted with rats dosed with nine azo dyes based on dimethyl-, dimethoxy-, or dichlorobenzidine to determine whether the free amine congeners, their monoacetyl or diacetyl metabolites, or alkaline hydrolyzable conjugates were excreted in the urine. After preliminary tests of the dyes, 2-mg doses were administered to each animal and urine samples were collected at intervals up to 96 hours. EC/GC procedures were based on the analysis of heptafluorobutyryl derivatives of the free amine congener moieties or their monoacetyl metabolites. Peak levels of metabolites were excreted either 0-12 or 12-24 hours after administration and, in seven of nine instances, no metabolites persisted in the urine after 48 hours. Minimum detectable levels of all metabolites were 12 ppb or less. All nine dyes were shown to be converted to measurable levels of their benzidine-congener-based metabolites in rats.

Direct Red 2 was given as an aqueous solution to rats and hamsters to determine whether the dye is cleaved to potentially carcinogenic aromatic amines. Sensitive and specific assays of the urine from treated animals by EC/GC revealed appreciable levels of 3,3'-dimethylbenzidine, mono- and di-acetyldimethylbenzidine, and alkaline hydrolyzable conjugates. Peak concentrations of the metabolites in urine occurred 12 to 24 hours after administration to rats, and within 12 hours in hamsters. The levels of all metabolites and conjugates diminished rapidly in both species after peak concentrations were reached, with no residues detected after 96 hours. The results conclusively demonstrated in vivo cleavage of the dye in both species, and it is proposed that the analytical methods employed can be used for chemical monitoring of human urine.

A simple and sensitive procedure is described for the quantitative determination of N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), the principal metabolite of the bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN-4), in urine. BCPN was analyzed by gas-liquid chromatography as its methyl ester after extraction with ethyl acetate from urine followed by esterification with diazomethane. Species and strain variations in susceptibility to bladder carcinogenesis induced by BHBN-4 are discussed in relation to the urinary excretions of BCPN, determined by the new procedure, after oral administration of the compound to rats (4 strains), mice, hamsters, and guinea pigs at a dose approximately equal to the daily dose used in chronic carcinogenicity assay.

A detailed study of the urinary metabolites of N'-nitrosonornicotine has been performed, employing a simple high pressure liquid chromatographic method. The percentage excretion of the principal urinary metabolites was determined over a dose range of 3-300 mg/kg in the F-344 rat, as follows: 4-hydroxy-4-(3-pyridyl)butyric acid (37.1-53.3%, respectively, of the dose), N'-nitrosonornicotine-I-N-oxide (6.7-10.7%), norcotinine (3.2-5.1%), 4-oxo-4-(3-pyridyl)butyric acid (31.1-12.8%), N'-nitrosonornicotine (3.3-5.2%). In the strain A mouse and Syrian golden hamster, the urinary metabolites were qualitatively similar to those observed in the F-344 rat. The interrelationships of the various metabolites of N'-nitrosonornicotine which have been observed in vitro and in vivo were established. The in vitro metabolites resulting from 2'-hydroxylation by liver microsomes, myosmine and 4-hydroxy-I-(3-pyridyl)-1-butanone were converted, by the F-344 rat, primarily to 4-oxo-4-(3-pyridyl)butyric acid as a urinary metabolite. The in vitro metabolite resulting from 5'-hydroxylation by liver microsomes, 2-hydroxy-5-(3-pyridyl)tetrahydrofuran, gave 4-hydroxy-4-(3-pyridyl)butyric acid as its major urinary metabolite, apparently via 5-(3-pyridyl)-tetrahydrofuran-2-one. N'-nitrosonornicotine-I-N-oxide, the remaining major in vitro metabolite, was excreted to a large extent unchanged in F-344 rat urine. The urinary metabolites from 2'-hydroxylation and 5'-hydroxylation of N'-nitrosonornicotine, 4-oxo-4-(3-pyridyl)butyric acid and 4-hydroxy-4-(3-pyridyl)butyric acid, respectively, were not formed from the in vivo metabolite norcotinine and were ot interconverted significantly by the F-344 rat. Thus, these metabolites appear to be reliable indicators for the two possible in vivo alpha-hydroxylations of N'-nitrosonornicotine.

Specific, precise and sensitive methods are described for determining traces of potentially carcinogenic metabolites of Direct Red 2 and Direct Blue 15 in rat and hamster urine and to monitor the urine from workers who may be occupationally exposed to these dyes. This methodology is generally applicable to metabolites of azo dyes based on benzidine or three of its congeners. The benzidine-congener free amines, their mono and diacetylated analogs and alkaline hydrolyzable conjugates were determined after appropriate extraction and hydrolysis by HPLC or EC/GC. Residues of metabolites in rat, hamster, and human urine were determined at levels as low as 1 ppb. Supplementary information is also presented concerning hydrolysis of diacetylated metabolites and the stability of Direct Red 2 and Direct Blue 15 in rat, hamster and human urine.

The bacterial bioassay procedure provides a sensitive test for the presence of mutagenic activity in urine. Its sensitivity for detecting the presence of individual chemicals may not be as high as that of specific analytical methods, but it has the following advantages: (1) many substances and metabolites may be active in a single assay, making it possible to detect mutagenic activity from unanticipated sources; (2) biological activity is demonstrated, rather than simply the presence of substances; or (3) the assay may also reflect the 'integrated' effect of multiple substances, although this capability has not been well characterized and (4) the assay can be easily coupled with a chemical analysis. The chief disadvantages of the test system are lack of sensitivity for certain specific substances, as compared to chemical techniques, and possible interference from substances normally present in urine (such as amino acids). The urinary mutagenicity assay is most useful when exposure to carcinogens and mutagens is suspected but when the specific chemical is unknown, when chemical analytical techniques are not available or when exposure is to undefined complex mixtures.

Analysis of 24 h urine samples of rats administered N-nitrosodipropylamine (NDPA), N-nitroso-2-hydroxypropylpropylamine (NHPPA) or N-nitroso-2-oxopropylpropylamine (NOPPA) showed that the major excretion product in each case was the beta-glucuronide of NHPPA. This conjugate was shown to be present at levels of about 5%, 80% and 50% of the administered dose for NDPA, NHPPA and NOPPA, respectively, based on exhaustive hydrolysis of the urine samples with beta-glucuronidase. Generally only low levels (less than 2%) of the unchanged nitrosamines or their unconjugated metabolites (NHPPA for administered NDPA and NHPPA for administered NOPPA) were detected. We were unable to detect N-nitrosomethylpropylamine or N-nitrosodimethylamine in the urine of any of the animals.

Metronidazole is a drug used for the treatment of trichomonal vaginitis, amebiasis, giardiasis, and certain anaerobic bacterial infections in humans. Acetamide and N-(2-hydroxyethyl)oxamic acid are metabolites of metronidazole in the rat, and we find small amounts of both metabolites in the urine of human patients taking the drug. Although acetamide is carcinogenic for rats, we do not believe that our finding further defines metronidazole's risk for humans. That risk can only be estimated from surveillance of people previously exposed to the drug.

The relationship between parasite infestation and chemical mutagen metabolism was investigated in this study. Schistosoma hematobium, long associated with increased incidence of bladder cancer in humans, was chosen as a model parasite. Urine samples, serum samples, and liver tissue extracts (S-9) from infested and control hamsters were used with the Ames Salmonella/microsome test to follow 3,3'-dichlorobenzidine (DCB), aflatoxin B1 (AFB1), and 2-acetylaminofluorene (AAF) mutagenicity. Liver S-9 preparations from infested and control hamsters showed little difference in activation potential for DCB and AFB1. Aroclor 1254-induced rat liver S-9, however, was remarkably efficient at reducing the mutagenicity of DCB. This process was reversible by beta-glucuronidase (BG). Studies on infested and control hamsters indicated increased BG activity in serum and urine. Urine concentrates (UC) from infested and control animals were not mutagenic by themselves, but did enhance the mutagenicity of AAF and DCB in the presence of S-9 and BG. Urine concentrates from infested animals showed greater enhancement of DCB mutagenicity than did UC from control animals. These data suggest that increased BG and unknown urinary factors in infested hamsters play a role in altering chemical mutagen activity.

Studies were undertaken to determine whether urine collected from rats during the 24 h following exposure to the colon carcinogen 1,2-dimethylhydrazine (DMH) contained substances which would induce specific antitumor immunity. The evaluation for such components was carried out by administering the test and control urine to young male adult Fisher F344 inbred rats and then establishing the animals' antitumor cell-mediated immunity (CMI) at a 14-day post-exposure interval. The CMI was measured by determining the injury and destruction inflicted upon cultured X-ray-induced rat small bowel adenocarcinoma target cells by peripheral blood lymphoid cells obtained from the animals exposed to the urine. A significantly increased CMI was found to be induced by the urine collected from the chemically exposed rats, thus suggesting the presence of mutagenic/carcinogenic components. In addition, exposure through the levels of CMI that were induced, with an approximate threshold detection limit of 100 microgram (1.7 mumol)/kg body weight (100 ppb) to the chemical. These preliminary findings suggest that such immune measurements might serve as a basis for the development of a rapid and inexpensive bioassay for monitoring population exposures to carcinogenic substances.

New metabolites of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent mutagen and carcinogen formed during cooking of meat or fish, have been identified and quantitated in the urine and bile of rats. Administration was either by a pulse gavage dose of 40 mg/kg [2-14C]IQ or by dietary intake of 300 ppm IQ for 6 weeks. The metabolites were isolated by high-performance liquid chromatography and quantitated by radioactivity. They were then characterized by their resistance or sensitivity to hydrolytic enzymes or acid hydrolysis, by nuclear magnetic resonance and mass spectrometry, or coinjection with a synthetic sample. A minor metabolite was the IQ N-glucuronide. A major metabolite was formed by hydroxylation of IQ at the 5-position; it was present in urine and bile and was conjugated as the glucuronide or sulfate ester, which together accounted for about 40% of urinary or biliary metabolites. The unconjugated compound partially adsorbs onto the high-performance liquid chromatographic columns used. The amounts of 5-OH-IQ present as conjugates in urine or bile were similar, irrespective of mode of administration. Thus, hydroxylation of IQ on carbon 5 followed by type II conjugation reactions yields quantitatively important metabolic products.

Rats and hamsters were administered a single dose of N-[1-14C]nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), and their urinary metabolites were examined at various time intervals. In both species, urinary excretion of radiolabeled metabolites reached a plateau at 6 h following injection. At this time, 35 and 28% of the total dose was found in the urine of rats and hamsters, respectively. Separation by liquid chromatography and subsequent characterization by nuclear magnetic resonance, gas chromatography-mass spectroscopy, and infrared showed that the major metabolites in rat urine were HPOP, N-nitrosobis(2-hydroxypropyl)amine (BHP), and their glucuronic acid conjugates. The conjugates accounted for 30 and 9%, while free HPOP and BHP accounted for 42 and 16% of the total metabolites, respectively. Hamster urine, on the other hand, contained free HPOP, BHP, their glucuronic acid conjugates, and a sulfate ester of HPOP not found in rat urine. Six h following administration of HPOP, hamster urine contained BHP, BHP glucuronide, HPOP, HPOP glucuronide, and HPOP sulfate ester at levels of 35, 9, 16, 9, and 14%, respectively. These data suggest that hamsters reduce HPOP to BHP more efficiently than rats, while rats are more effective in forming their glucuronic acid conjugates. Hamsters differ significantly from rats in their capacity to form and excrete the sulfate ester of HPOP.

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT greater than nitrofurantoin greater than 5-nitro-2-furamidoxime greater than 5-nitrofurfurylidene diacetate greater than 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with beta-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.

The metabolic fate of N, N-dipropylnitrosamine (DPN) and N, N-dipentyl-(=diamyl) nitrosamine (DAN), which induced tumors principally in the liver and esophagus but not in the urinary bladder, was studied in the rat, in order to elucidate the reason for this lack of carcinogenicity to the urinary bladder in contrast with the butyl homolog, N, N-dibutylnitrosamine (DBN), a potent bladder carcinogen. The principal urinary metabolite of DPN was identified as the omega-oxidation product N-propyl-N-(2-carboxyethyl) nitrosamine. The (omega-1)-oxidation product N-propyl-N-(2-hydroxypropyl) nitrosamine and its glucuronic acid conjugate were also characterized as metabolites. The principal urinary metabolite of DAN was N-amyl-N-(2-carboxyethyl) nitrosamine, which was formed by metabolic shortening of the amyl chain by omega- and beta-oxidations according to the Knoop mechanism. Besides this, seven compounds oxidized at omega-, (omega-1)-, (omega-2)-, and (omega-3)- positions of one or both amyl chains were isolated and characterized, some of which were shown to be also present as the corresponding glucuronide. The metabolic pattern of DPN and DAN was compared with that of DBN, and the lack of carcinogenicity of the former N-nitrosamines to the urinary bladder of rats is discussed on the basis of their urinary metabolites.

The metabolic fate of two urinary bladder carcinogens, N-butyl-N-(4-hydroxy-butyl) nitrosamine (BBN) and N-N-dibutylnitrosamine (DBN), was studied in the guinea pig, in order to elucidate species differences of response to these N-nitrosamines in this animal species and the rat. Based on the urinary metabolites characterized after oral administration of these compounds, the metabolic pathways of BBN and DBN in the guinea pig were shown to be essentially similar to those in the rat. The principal urinary metabolite of BBN and DBN in the guinea pig, however, was not N-butyl-N-(3-carboxypropyl) nitrosamine (BCPN), as was the case in the rat, but the glucuronic acid conjugate of BBN and that of N-butyl-N-(3-hydroxybutyl) nitrosamine, respectively. The species variation in response to BBN and DBN as bladder carcinogens in these animals is discussed on the basis of the urinary excretion of BCPN.

Six batches of food traditionally and commonly consumed by southern Chinese, including two samples of dried shrimps and four samples of different species of salted fish, were tested for mutagenic properties using Salmonella typhimurium TA98 and TA1000. Mutagenic activities toward both tester strains were found in all preparations. In most cases, these activities were enhanced by liver microsomal activation. Urine collected from experimental rats regularly fed salted fish also showed mutagenic activity. The level of this activity decreased markedly when the experimental rats were transferred from a salted fish diet to Purina rat chow. Our data suggested the presence of mutagenic/carcinogenic substances in some local preserved foods and at least one of them, salted fish, has been suspected on epidemiological and experimental evidence to be a possible co-carcinogenic factor in the development of nasopharyngeal carcinoma in southern Chinese. Our finding is compatible with this hypothesis.

The kinetics of distribution of radiolabelled [2-14C]IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and [2-14C]MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) following the oral administration to BALB/c mice of single doses were studied. Both compounds were taken up into the blood-stream and other tissues rapidly after administration and approximately 20-25% of the radioactive dose of IQ or MeIQx was excreted in urine over 6 h, reflecting the rapid absorption of the mutagens. Significantly greater levels of MeIQx than IQ were isolated from the lungs and blood of treated mice. In studies of the uptake of IQ from closed sections of the gut, little IQ was absorbed from the stomach. Although there was some evidence that it could be absorbed from the large intestine, the primary site of IQ absorption was the small intestine.

Rats exposed to technical grade dinitrotoluene (DNT) develop hepatocellular carcinomas. Humans may be exposed to DNT during its manufacture and use. To permit comparisons of human excretion patterns of DNT metabolites with those previously observed in rats, urine specimens were collected over a 72-hr period from workers at a DNT manufacturing plant. Samples were analyzed for 2,4- and 2,6-DNT and putative metabolites by gas chromatography-mass spectrometry. Urine from workers exposed to DNT contained 2,4- and 2,6-DNT, 2,4- and 2,6-dinitrobenzoic acid, 2,4- and 2,6-dinitrobenzyl glucuronide, 2-amino-4-nitrobenzoic acid, and 2-(N-acetyl)amino-4-nitrobenzoic acid. Excretion of these metabolites peaked near the end of the workshift, but declined to either very low or undetectable concentrations by the start of work the following day. The calculated half-times for elimination of total DNT-related material detected in urine ranged from 1.0 to 2.7 hr, and those of individual metabolites from 0.8 to 4.5 hr. The most abundant metabolites were 2,4-dinitrobenzoic acid and 2-amino-4-nitrobenzoic acid, collectively accounting for 74 to 86% of the DNT metabolites detected. The data indicate that urinary metabolites of DNT in humans are qualitatively similar to those found in rats, but quantitative differences exist in the relative amounts of each metabolite excreted.

The hepatic metabolism of arylamine bladder carcinogens to N-hydroxy arylamine N-glucuronides, their excretion in the urine, and their subsequent acidic hydrolysis to highly carcinogenic and reactive N-hydroxy arylamines have been proposed as essential steps in arylamine-induced urinary bladder carcinogenesis. In this study, alteration of urinary pH, inhibition of metabolic sulfation, and blockage of biliary disposition were shown to profoundly affect the urinary excretion of the probable ultimate bladder carcinogen, N-hydroxy-2-naphthylamine (N-HO-2-NA) and its N-glucuronide conjugate. The normal pH of rat urine (6.7) was altered to 5.7 or 7.7 by administration of NH4Cl or NaHCO3 in the drinking water. Subsequent treatment with either 2-naphthylamine (2-NA) or 2-nitronaphthalene (2-NN) resulted in increased urinary levels of free N-HO-2-NA (relative to its N-glucuronide) in acidic urines and decreased relative amounts of free N-HO-2-NA in alkaline urines. In addition, 2-NN yielded 5--10-fold greater levels of urinary N-HO-2-NA and its N-glucuronide than rats given 2-NA; and 2-NA was not detected as a urinary metabolite of 2-NN. Some 12 additional metabolites of 2-NA and 2-NN were also found. Of these, 2-amino-1-naphthol and its sulfate and glucuronide conjugates were quantitated. From these data, 2-NA and 2-NN appear to share common metabolic pathways which yield free N-HO-2-NA as a putative ultimate urinary bladder carcinogen. Pentachlorophenol, a known inhibitor of hepatic sulfotransferases, was shown to cause a 2--3-fold increase in the urinary levels of N-HO-2-NA N-glucuronide and N-HO-2-NA from 2-NA-treated rats. Similarly, inhibition of the biliary excretion of 2-NA by bile duct ligation resulted in a 6-fold increase in total urinary N-HO-2-NfA. Furthermore, analyses of bile revealed that substantial amounts of N-HO-2-NA N-glucuronide, but not free N-HO-2-NA, were present. The role of urinary versus biliary excretion of N-hydroxy arylamines in relation to bladder and colon carcinogenesis is discussed.

A series of experiments has indicated that the bladder surface is lined with a sulfated polysaccharide that is capable of acting as a nonspecific antiadherence factor. It is capable of preventing the interaction of the transitional cells with many substances in urine, including bacteria, calcium, protein, and potential carcinogens. This interface of urine and transitional epithelium may be clinically important in many urologic disease states, since a malfunction of this layer could be involved in a spectrum of urologic diseases that includes irritable bladder syndromes (e.g., detrusor instability, interstitial cystitis, urethritis, radiation cystitis, cyclophosphamide-induced cystitis), carcinoma in situ, calculus disease, infections, and even carcinogenesis. It is important to point out that these investigations have shown that the natural surface mucus can be replaced by any of several substitutes that are as capable of preventing adherence as the natural glycosaminoglycans. One such substance, pentosanpolysulfate, is available in an oral form that is excreted in urine. To date, it has been used to successfully treat one disease of the urinary bladder, interstitial cystitis.

Analytical chemical procedures are described for determining traces of possible metabolites of two azo compounds. Direct Black 38 and Pigment Yellow 12, in hamster urine and to monitor the urine from workers who may be occupationally exposed during the manufacture or use of the dye and pigment. These methods were required for metabolism studies designed to assess the hazards that may occur if the two compounds are converted by in vivo mechanisms to potential carcinogens. Salient elements of the procedure are: extraction of the free aromatic amines and neutral compounds; alkaline hydrolysis of the aqueous phase and extraction of any hydrolyzed conjugates as free amines, and the analysis of the free amines and acetylated metabolites directly by high pressure liquid chromatography or by electron capture gas chromatography after conversion of the amines to heptafluorobutyryl derivatives. Residues of metabolites in hamster and human urine were determined at levels as low as 1 ppb. Ancillary data concerning hydrolysis of diacetylated metabolites and partition values for possible metabolites in various solvent systems are also presented.

We assessed the possibility to detect promoters of bladder carcinogenesis by a short-term in vitro assay utilizing the activation of Epstein-Barr virus (EBV) expression in EBV genome-carrying human lymphoblastoid cells. This system is composed of EBV-nonproducer Raji cells as the indicator, n-butyrate as the EBV-inducer and the test substance. None of eight known bladder carcinogens (2-naphthylamine, 2-acetylaminofluorene, N-butyl-N-(4-hydroxybutyl)nitrosamine, N-butyl-N-(3-carboxypropyl) nitrosamine, N-methyl-N-nitrosourea, N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide, benzidine, cyclophosphamide) or five promoters (sodium saccharin, sodium cyclamate, urea, allopurinol, DL-tryptophan) were detected by this system. Normal urine from 20 rats did not activate the EBV genome. Neither the 0.45 micron membrane filtrates nor ether extracts of urine obtained from rats given one of 4 bladder carcinogens, 4 promoters or phorbol 12-myristate 13-acetate reacted with this system, whereas those from rats given either vincristine or vinblastine did, although their activity for promoting bladder carcinogenesis is not known. Urine samples from 42 patients with bladder cancers, when tested as filtrates, ether extracts or XAD-2 resin adsorbates, did not activate EBV expression. These results suggest that EBV-activating substances contribute little to promotion of bladder carcinogenesis and that this test might not be applicable for screening of tumor promoters in urine.

Major metabolites, isolated from rat bile and urine after administration of a single dose of 7-methoxy-2-nitronaphtho[2,1-b]furan (R 7000; MNNF) labelled with 14C on the furan ring and on the methoxy group, were identified by comparison of their chromatographic behaviour and mass spectra with synthetic authentic reference compounds. Analysis of metabolites indicated three metabolic pathways for this compound in vivo, namely, demethylation of the methoxy group, hydroxylation of the aromatic ring and cleavage of the furan ring, followed by the reduction of the nitro group to amine.

Two additional urinary metabolites of the chemical oncogen 3-hydroxyxanthine are now identified as 8-chloroxanthine and 8-methylmercaptoxanthine. Such products are thought to be derived from a reactive intermediate which can be tentatively considered to be a proximate oncogen. Since each of these 8-substituted xanthines has also been obtained in vitro by reactions of 3-acetoxyxanthine with chloride ion or methionine, their production in vivo can be explained as resulting through the metabolic formation of an activated ester with a reactivity similar to that of the chemical model.