Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages.

L-Arginine-derived nitric oxide (NO) and its derivatives, such as peroxynitrite and nitrogen dioxide, play a role in inflammation and also possibly in the multistage process of carcinogenesis. We investigated the effect of various non-steroidal anti-inflammatory agents and related compounds on the induction of NO synthase (NOS) in RAW 264.7 macrophages activated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Low concentrations of curcumin, a potent anti-tumour agent having anti-inflammatory and anti-oxidant properties, inhibited NO production, as measured by the amount of nitrite released into the culture medium in 24 h (IC50 = 6 microM). NOS activity in soluble extracts of macrophages activated for 6-24 h in the presence of curcumin (10 microM) was significantly lower than that of macrophages activated without curcumin. Northern-blot and immunoblotting analyses demonstrated that significantly reduced levels of the mRNA and 130-kDa protein of inducible NOS were expressed in macrophages activated with curcumin, compared to those without curcumin. Inhibition of NOS induction was maximal when curcumin was added together with LPS and IFN-gamma and decreased progressively as the interval between curcumin and LPS/IFN-gamma was increased to 18 h.

Increased nitrosamine and nitrate biosynthesis mediated by nitric oxide synthase induced in hamsters infected with liver fluke (Opisthorchis viverrini).

We previously reported that increased endogenous nitrosation in human subjects infected with the liver fluke Opisthorchis viverrini in north-east Thailand could be a risk factor for the development of cholangiocarcinoma. In the present study we examined our hypothesis that this increased endogenous nitrosation is mediated by nitric oxide (NO) synthase induced by O. viverrini infestation. Syrian golden hamsters experimentally infected with O. viverrini liver fluke excreted in the urine significantly greater amounts of nitrate, a stable oxidization product of NO, than untreated hamsters (3.64 +/- 0.86 versus 2.64 +/- 0.60 mumol/hamster/day, P < 0.001). When the rapidly nitrosatable thiazolidine 4-carboxylic acid was administered orally, the infected hamsters also excreted significantly elevated levels of N-nitrosothiazolidine 4-carboxylic acid than untreated hamsters (4.27 +/- 2.20 versus 2.33 +/- 1.13 nmol/hamster/day, P < 0.01), indicating that endogenous nitrosation is elevated in the animals with liver fluke. NO synthase activity measured in liver cytosol was about twice as high in the infected hamsters as in untreated animals. The enzyme, whose biochemical characteristics were similar to that induced in activated murine macrophages, was immunohistochemically localized in the cytoplasm of macrophages and eosinophils in the inflammation zone surrounding the parasite-containing bile ducts. These results support our hypothesis that, in fluke-infected subjects, NO synthase induction leads to excess production of NO and the observed elevated endogenous nitrosation. Since high concentrations of NO exert cytotoxic and mutagenic effects per se, excess NO produced in chronically infected/inflamed tissues may also play a role in initiation and subsequent modulation stages of cholangiocarcinoma development.

Urinary excretion of nitrosamino acids and nitrate by inhabitants of high- and low-risk areas for nasopharyngeal carcinoma in southern China.

The hypothesis that endogenous synthesis of nitrosamines from dietary precursors is a risk factor for nasopharyngeal carcinoma (NPC) in China was tested by applying the nitrosoproline (NPRO) test to subjects living in high- and low-risk districts for NPC in Zangwu county, Guangxi region, in southern China. Samples of 12-h urine were collected from 77 subjects: (a) before any treatment; (b) after ingestion of proline; and (c) after ingestion of proline together with vitamin C. NPRO, other nitrosamino acids, and nitrate were measured as indices of exposure to preformed and endogenously formed nitrosamines or their precursors. The NPRO level after proline intake was significantly increased in subjects from the high-risk area (P = 0.012) and markedly reduced after ingestion of ascorbic acid (P = 0.007), but such an effect was not seen in subjects from the low-risk area. Levels of N-nitrosothiazolidine-4-carboxylic acid and the sum of nitrosamino acids in subjects in the high-risk area were significantly reduced by ascorbic acid (P < 0.01) but were not reduced in subjects from the low-risk area. The urinary nitrate level was about twice as high in subjects from the high-risk area. In subjects from high- and low-risk areas combined, NPRO levels in any of the three dose groups were highly correlated with nitrate levels (P = 0.0001). These results demonstrate a higher potential for endogenous nitrosation in subjects living in the high-risk area of NPC and suggest the occurrence of nitrosation inhibitors in the diet consumed in the low-risk area.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of an inducible form of nitric oxide synthase in various organs of rats treated with Propionibacterium acnes and lipopolysaccharide.

Immunohistochemical localization of an endotoxin-inducible form of nitric oxide synthase was examined using rabbit polyclonal antibody against the enzyme purified from rat liver. In rats treated with Propionibacterium acnes and lipopolysaccharide, immunostaining was observed in macrophages, occasional lymphocytes, neutrophils and eosinophils in red pulp of spleen, Kupffer cells, endothelial cells and hepatocytes in liver, alveolar macrophages in lung, macrophages and endothelial cells in adrenal glands, and histiocytes, eosinophils, mast cells and endothelial cells in colon. Immunoreactivity was also evident in the following tissues: histiocytes and endothelial cells in kidney; histiocytes and neutrophils in esophagus; macrophages and eosinophils in duodenum; macrophages, some lymphocytes and mast cells in ileum; histiocytes in thymus; and endothelial cells in heart and aorta. Immunoreactivity was not detected in these organs from untreated rats. Positively staining cells in these rat organs appeared within 2.5 h after lipopolysaccharide administration; their number dramatically increased within the next 2.5 h, remained at high levels for a further 19 h, and then decreased over the following 24 h. The number of positive cells appearing correlated well with the nitric oxide synthase activity biochemically determined in the same organs.

Increased endogenous N-nitrosamine and nitrate formation by induction of nitric oxide synthase in rats with acute hepatic injury caused by Propionibacterium acnes and lipopolysaccharide administration.

In rats treated i.v. with heat-killed Propionibacterium acnes (100 mg/kg body wt), followed 5 days later by an i.v. dose of Escherichia coli lipopolysaccharide (LPS, 1 mg/kg body wt), acute hepatic cell necrosis was accompanied by significant induction of nitric oxide (NO) synthase activity in the liver. Endogenous nitrosation of thiazolidine 4-carboxylic acid (TCA, 50 mumol/rat) administered by three different routes (i.v., i.p. and p.o.) 5 h after LPS injection to the P. acnes-treated rats was assessed by analysing its nitrosated product (NTCA) excreted in 24 h urine. The amounts of NTCA formed in vivo after i.v., i.p. and p.o. administration of TCA were 4.07 +/- 1.00, 5.79 +/- 2.15 and 58.3 +/- 20.7 nmol/rat (n = 5-10) respectively, which were about 5-, 10- and 8-fold greater than those excreted by rats which had not been treated with P.acnes and LPS but received TCA by the same route. Nitrate concentration in plasma and NO synthase activity in the liver started to increase within 2.5 h after LPS injection, reached a maximum at 7.5 h and remained at high levels for several further hours. Levels of nitrite and nitrate in gastric contents were also increased significantly after LPS administration. The co-administration of N omega-nitro-L-arginine (an inhibitor of NO synthase) and LPS resulted in a marked reduction of urinary levels of nitrate and NTCA, indicating that nitrosation is mediated by NO synthase. These results together suggest that induction of NO synthase by infection with bacteria, parasite and viruses could result in increased endogenous nitrosation not only in the infected tissues but also in the stomach, where nitrosamines would be formed more rapidly under acidic conditions.

Polyclonal antibody against an inducible form of nitric oxide synthase purified from the liver of rats treated with Propionibacterium acnes and lipopolysaccharide.

A polyclonal antibody was raised in the rabbit against an inducible form of nitric oxide (NO) synthase (EC 1.14.23) purified from the liver of rats with acute liver necrosis induced by i.v. administration of Propionibacterium acnes and lipopolysaccharide. The antibody immunoprecipitated NO synthase activities in the soluble extract of the liver from treated rats. Western blot analysis showed that the cytosols of the liver, lung and spleen from the treated rats but not from non-treated rats, and that of murine macrophages cultured in the presence of lipopolysaccharide and interferon-gamma, contained immunoreactive protein with a molecular weight of 125 kDa. The antibody, however, does not cross-react with a 150 kDa constitutive form of NO synthase present in the brain of rats, indicating that the inducible and constitutive enzymes are immunologically distinguishable.

Carcinogenic tobacco-specific nitrosamines are present at unusually high levels in the saliva of oral snuff users in Sudan.

Exposure to tobacco-specific nitrosamines (TSNA) has been measured in the saliva of 12 users of Sudanese oral snuff (toombak). Using GC coupled to thermal energy analysis, levels of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were measured before, during and after snuff taking. In addition, two TSNA, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL), were detected in the saliva of tobacco chewers for the first time and were confirmed by GC-MS. Nine out of 10 subjects had detectable saliva levels of total TSNA before chewing (0.01-1.0 micrograms/ml) and immediately following chewing (0.1-2.6 micrograms/ml). During dipping, TSNA concentrations reached microgram/ml levels; (range; number of subjects positive) NNN: (0.6-2.1; 12/12), NAT (0.06-0.5; 2/12), NAB (0.05-1.9; 12/12), NNK (0.06-6.7; 11/12), NNAL (0.05-3.3; 11/12) and iso-NNAL (0.07-0.4; 8/12). These saliva TSNA levels, which are 10-100 times the levels previously reported, are consistent with recent observations of unusually high TSNA levels in Sudanese toombak. As several of these TSNA have been shown to be carcinogenic in animals and epidemiological studies have associated human snuff use with tumours of the oral cavity, these findings draw attention to a significant potential public health hazard.

Opisthorchis viverrini infestation and endogenous nitrosamines as risk factors for cholangiocarcinoma in Thailand.

Cholangiocarcinoma (CCA) is one of the most common cancers in north-east Thailand and has been associated with infestation by the liver fluke Opisthorchis viverrini (OV). Two samples of 12-hr overnight urine (after dosing with proline and ascorbic acid or proline alone) were collected from 20 inhabitants from each of 5 contrasting incidence areas for CCA. The incidence of CCA was not correlated with either the amount of NPRO or other nitrosamino acids, endogenous nitrosation potential (difference in NPRO levels between proline dose and proline and ascorbic dose), or nitrate level. However, when urinary levels of nitrosamino acids were compared in subjects living in high-risk areas, subjects who were positive for OV antibody excreted significantly more (p less than 0.01) NPRO (12.3 +/- 18.7 micrograms/12 hr) after proline ingestion than those who were negative (3.5 +/- 3.2 micrograms/12 hr). After ingestion of ascorbic acid, the NPRO levels in the positive subjects were significantly reduced (p less than 0.01) to 2.4 +/- 2.0 micrograms/12 hr, suggesting that endogenous nitrosation of proline was inhibited. Thus, endogenous nitrosation potential estimated from the difference between NPRO and the sum of nitrosamino acids excreted in the 2 urine samples was significantly higher in subjects positive for the OV antibody. Small amounts of pre-formed nitrosamines were found in fermented fish and pork food items, which are consumed frequently in the high-risk area for CCA. These results suggest that the interaction between chemical carcinogens, especially nitrosamines, and OV infestation may play a role in the development of cholangiocarcinoma in Thailand.

Unusually high levels of carcinogenic tobacco-specific nitrosamines in Sudan snuff (toombak).

The aim of these studies was to determine the levels of carcinogenic tobacco specific nitrosamines (TSNA) in Sudanese oral snuff (toombak) as recent retrospective epidemiological studies suggested an association between the use of toombak and subsequent development of oral cancer. We have analyzed the TSNA levels in 20 samples of Sudanese toombak, of four different quality levels, collected from five different vendors. Using GC coupled with thermal energy analysis, four TSNA were quantified in snuff extracts: N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Unusually high levels of these TSNA (mean; range, mg/g snuff, dry wt) were detected; NNN (1.13; 0.50-3.08); NAT (0.08; 0.02-0.29); NAB (0.22; 0.02-2.37); and NNK (2.31; 0.62-7.87). Previously, the highest levels of NNN and NNK reported in any snuff were 0.154 and 0.014 mg/g dry wt respectively. In comparison, the levels in Sudanese toombak were up to 20 and 560 times higher respectively. As the public health implications of these findings are significant, attempts should be made to reduce exposure to TSNA in oral snuff users in Sudan.

Endogenous nitrosamines and liver fluke as risk factors for cholangiocarcinoma in Thailand.

Cholangiocarcinoma (CCA) is one of the most prevalent cancers in north-east Thailand and has been associated with infestation by the liver fluke Opisthorchis viverrini (OV). Two samples of 12-h overnight urine (after dosing with 500 mg proline and 200 mg ascorbic acid or 500 mg proline alone) were collected from about 100 inhabitants in five contrasting incidence areas for CCA and hepatocellular carcinoma. The incidences of CCA and hepatocellular carcinoma were not correlated with either the amount of NPRO or other nitrosamino acids, endogenous nitrosation potential (difference in NPRO levels between proline dose and proline and ascorbic acid dose), or nitrate level. However, when urinary levels of nitrosamino acids were compared in subjects living in high-risk areas, subjects who were positive for OV antibody excreted significantly more (p less than 0.01) NPRO (12.3 +/- 18.7 micrograms/12 h) after proline ingestion than those who were negative 3.5 +/- 3.2 micrograms/12 h). After ingestion of ascorbic acid, the NPRO levels in the positive subjects were significantly reduced (p less than 0.01) to 2.4 +/- 2.0 micrograms/12 h, suggesting that endogenous nitrosation of proline was inhibited. Thus, endogenous nitrosation potential estimated from the difference of NPRO and sum of nitrosamino acids excreted in the two urine samples was significantly higher in subjects positive for the OV antibody. In addition, of the representative food samples and beverages consumed frequently in high-risk areas for CCA, fermented fish and pork contained N-nitrosodimethylamine (0-26 micrograms/kg), N-nitrosopyrrolidine (0-117 micrograms/kg) and N-nitrosopiperidine (0-23 micrograms/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrotyrosine as a new marker for endogenous nitrosation and nitration.

A sensitive and selective method has been developed for analysing 3-nitrotyrosine (NTTYR), an exposure marker for exogenous and endogenous nitrosating or nitrating agents, in tissue and blood proteins by gas chromatography-thermal energy analysis. Using this method, a number of kinetic studies were carried out. Free and protein-bound tyrosine were reacted easily to yield NTTYR. The method was also applied to the study of NTTYR formation in vivo; a dose-dependent increase in NTTYR was seen in both plasma proteins and haemoglobin obtained from rats 24 h after intraperitoneal injection of various doses (0.5-2.5 mumol/rat) of tetranitromethane. Major urinary metabolites of NTTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid. About 44% and 5% of the oral dose of NTTYR (100 micrograms/rat), respectively, was excreted as these metabolites. Some human urine samples were analysed for NHPA by gas chromatography-thermal energy analysis after ethyl acetate extraction and high-performance liquid chromatography purification; 2.8 +/- 2.3 (mean +/- SD; n = 11) micrograms/24 h, ranging from 0-7.9 micrograms/24 h, were detected (detection limit, 0.2 micrograms/l). In conclusion, NTTYR in proteins or its metabolites in urine could be readily analysed by gas chromatography-thermal energy analysis as a new, additional marker for endogenous nitrosation and nitration.

Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins.

3-Nitrotyrosine (NTYR) in tissue or blood proteins was evaluated as a possible exposure marker for exogenous and endogenous nitrosating or nitrating agents. A sensitive and selective method for analysing NTYR by gas chromatography with a thermal energy analyser (GC-TEA) was developed. Using this method, a number of kinetic studies were carried out. It was found that free and protein-bound tyrosine residues easily react with nitrating/nitrosating agents to yield NTYR. NTYR formation in vivo showed a dose-dependent increase in NTYR in both plasma proteins and haemoglobin obtained from rats 24 hr after ip injection of various doses (0.5-2.5 mumol/rat) of tetranitromethane. Major urinary metabolites of NTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid (NHPL). About 44% and 5% of the oral dose of NTYR (100 micrograms/rat) was excreted as NHPA and NHPL, respectively. Eleven 24-hr human urine samples were analysed for NHPA by GC-TEA after ethyl acetate extraction and HPLC purification: quantities ranging from 0 to 7.9 micrograms/24 hr, mean +/- SD 2.8 +/- 2.3 (n = 11) were detected (detection limit 0.2 micrograms/litre). NTYR in proteins or its metabolites in urine can be readily analysed by GC-TEA as a new/additional marker for endogenous nitrosation and nitration.

Modulating effects in human diets of dietary fibre and beef, and of time and dose on the reactive microcapsule trapping of benzo[a]pyrene metabolites in the rat gastrointestinal tract.

Trapping by magnetic polyethyleneimine (PEI) microcapsules was utilized to investigate the influence in male rats of dose, human dietary composition and time-dependence on reactive metabolites of benzo[a]pyrene (B[a]P) in the gastrointestinal (GI) tract; also, PEI microcapsules modified with copper phthalocyanine tetrasulphonic acid (CPTS) were tested in vivo for trapping of endogenous mutagens having planar molecular structure. In a preliminary experiment the PEI microcapsules were administered by gavage at 0, 24 and 48 h, with [14C]B[a]P at 2 h to chow-fed BDVI rats; microcapsules were recovered from faeces collected at 24, 48 and 72 h, and then subjected to an extraction sequence showing that the trapped B[a]P metabolites were inconsistent with B[a]P diol epoxide trapping (as previously found) and unaltered by elapsed time or 5-fold dose alteration of B[a]P. Then five groups of F344 rats were fed isocalorically either one of four low-fat human diets or rat chow; in order to investigate influences of diet both on B[a]P and endogenous mutagens, half of each group was tested at 2 weeks with this PEI microcapsule/[14C]B[a]P protocol and then at 3 weeks, PEI-CPTS microcapsules (two gavages). So as to provide a cross-over comparison, the other half of each group was first tested with PEI-CPTS microcapsules followed by PEI microcapsules/[14C]B[a]P 1 week later. The human diets were prepared from cooked British foods so as to simulate the adequate intake of all nutrients required by humans; but with 3-fold differences in intake levels of beef and dietary fibre non-starch polysaccharide (NSP), while ensuring the same intake of available energy, protein, fat and calcium. They gave very similar body-weight gains in the four groups but greatly reduced faecal weight, protein and total faecal enzyme activity compared with chow; the extraction pattern of microcapsule-trapped B[a]P metabolite radioactivity was not significantly altered. However, human diet consumption caused a 2- to 6-fold increase in B[a]P metabolite binding to microcapsules and reductions in microcapsule recovery, net 70-h B[a]P excretion, faecal protein and total activities for beta-glucuronidase and beta-galactosidase; these effects were more pronounced after 3 weeks, presumably due to prolonged dietary adaptation. Increased NSP in human diets significantly increased the B[a]P metabolite excretion and marginally reduced the microcapsule binding. The increase in microcapsule binding of B[a]P metabolites, interpreted as reflecting an increased amount of reactive metabolites encountered, was related to the dietary intake weight ratio of beef/NSP.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of direct-acting genotoxic substances in nitrosated smoked fish and meat products: identification of simple phenolic precursors and phenyldiazonium ions as reactive products.

Epidemiological studies have associated the consumption of smoked fish and meat products with an increased risk of stomach cancer. Therefore, the reaction of such smoked foods with nitrite under acidic conditions was investigated and was shown to produce potent direct-acting genotoxic substances as detected by the SOS Chromotest. Similar genotoxic activity was observed in nitrosated samples of wood-smoke condensates. Simple phenolic compounds such as phenol, 3-methoxycatechol, catechol and vanillin were identified as the precursors of the genotoxic substances. These phenolic compounds also exhibited direct-acting genotoxicity after nitrosation. The major genotoxic substances formed after nitrosation of phenol were isolated and identified as 4- and 2-hydroxyphenyldiazonium ions. Nitrosation of various wood-smoke condensates was found to generate the same type of diazonium compounds, which in part account for the genotoxicity of nitrosated smoked foods.

Recoverable, semipermeable, microencapsulated DNA surrogates for monitoring the colorectal cavity; in-situ effects of fibre and meat in human diets on benzo[a]pyrene and possible endogenous cross-linking agents.

Semipermeable magnetic microcapsules containing polyethyleneimine (PEI) as a DNA surrogate are shown to trap 14C-benzo[a]pyrene and hitherto unknown, endogenous, putative cross-linking agent(s) within the gut of male Fischer rats. Trapping is substantially modulated by complete, cooked human diets fed isocalorically and varied three-fold in either beef, fat or bran fibre nonstarch polysaccharide within the normal human intake levels. Preliminary results indicate that the crosslinking agent(s) are derived from microflora. Using metabolized benzo[a]pyrene as a model DNA damaging agent within the gut, beef and decreased bran fibre were found to increase its availability, paralleling risk alterations found in nutritional epidemiology. These novel microcapsules are capable of intercepting a range of substances relevant to DNA damage.

Magnetic semi-permeable polyethyleneimine microcapsules for monitoring of N-nitrosation in the gastrointestinal tract.

Semi-permeable polyethyleneimine (PEI) microcapsules were developed recently for trapping unstable electrophilic products in the gastrointestinal tract. Their N-nitrosation has been investigated in vitro and in vivo in the present study. At acid pH N-nitrosation was found to be linearly dependent on nitrite concentration, without a pH maximum. Up to 70% of the nitrosating agent was converted to N-nitroso products that were retained in the microcapsules and detected by a total N-nitroso assay procedure. Microcapsules prepared with different formulations (in order to vary membrane characteristics) provided a limit of detection in the range 1-10 nmol N-nitrosating agent and were also found to have a different capacity for N-nitrosation. Based on n.m.r. data it seems that such N-nitrosation is favoured by the incomplete protonation of this polyamine at acid pH and that nitrosation occurs at the least hindered secondary amine functions. Microcapsules were administered intragastrically in doses of 2 or 6 million to rats also receiving nitrite in the drinking water and were recovered magnetically from faeces. Relative to the maximum possible yield of N-nitrosated microcapsules that could have been excreted within the first 24 h, the excretion (maximum 4.9%) was found to depend on number of capsules administered and the time of administration relative to access to nitrite. Although performed with only a few animals, these preliminary data indicate a performance possibly superior to existing endogenous nitrosation indicators, and a dual purpose system to trap both nitrosating agents and their direct-acting DNA-damaging N-nitroso products.

Binding of benzo[a]pyrene metabolites in the rat intestinal lumen by magnetic polyethyleneimine microcapsules following an intragastric dose of [14C]benzo[a]pyrene.

Semi-permeable magnetic polyethyleneimine (PEI) microcapsules have been developed to trap carcinogens and their metabolites in vivo and their time-dependent binding of a model carcinogen, [14C]benzo[a]pyrene [( 14C]BaP), is studied within the intestinal lumen. Overall, approximately 0.5% of an intragastric BaP dose was bound by these microcapsules recovered from faeces with specific binding of metabolites (nmol/10(6) recovered microcapsules) being similar in the 0-24-h and 24-48-h periods, but approximately 10-fold lower in the 48-72-h period. Successive extractions of microcapsules with ammoniacal methanol, 2.5 N HCl, methanol and dimethylsulfoxide released approximately 60% of bound radiolabeled and the unextracted radiolabel was presumed to have been bound covalently. By contrast, greater than 90% of bound radiolabel was extractable from the faeces of the treated animals and from microcapsules treated in vitro with [14C]7,8-dihydroxy-9,10-epoxytetrahydrobenzo[a]pyrene (BaPDE), indicating that the in vivo microcapsule-bound metabolites were not derived either from adsorbed faecal material or from [14C]BaPDE formed in situ. A time-dependent appearance of BaP 3,6-dione was found. Also the qualitative and quantitative patterns of metabolites trapped by microcapsules, as assayed by h.p.l.c., were consistent only with a unique set of BaP metabolites being bound within the intestinal lumen. Hence these carcinogen-binding microcapsules can be used to investigate the in situ formation of carcinogen metabolites within the intestinal tract.

Trapping of chemical carcinogens with magnetic polyethyleneimine microcapsules: III. In vivo trapping of electrophiles from N-methyl-N-nitrosourea and recovery from feces.

The use of semipermeable magnetic polyethyleneimine (PEI) microcapsules for trapping carcinogens in vivo is described. After intragastric administration to rodents, up to 40% of the microcapsules were extracted magnetically from fecal suspensions, with most recovered in the first 24 h after administration. The mean diameter of the recovered microcapsule population was in some cases larger than that administered. The changes in size distribution and incomplete recovery could be ascribed to the magnetic extraction technique rather than loss or destruction of microcapsules in vivo. By contrast, magnetic hemoglobin microcapsules were not stable in vivo and were recovered in very low yields. An important factor determining the recovery of administered PEI microcapsules was the amount of encapsulated magnetite. Microcapsules administered intragastrically to rodents trapped up to 0.02% of an intragastric dose of N-[methyl-14C]-N-nitrosourea (1). Binding was dose dependent with the limiting factor being the number of available binding sites; increasing the number of administered microcapsules accordingly increased the total amount of 1 bound by them. After administration of [14C--CH3]-derivatized microcapsules, the recovery from feces of microcapsule-associated radioactivity was 48-74%, up to twice the numerical recovery. Although this indicates that microencapsulated labeled core PEI was recovered, it could not be released upon sonication. This was due in part to an increased resistance of the microcapsules to sonication caused by GI tract transit that was probably due to nonspecific absorption of substances. Subsequent acid treatment released some radiolabeled core PEI, as indicated by precipitation with polyacrylic acid. Excreted microcapsules were found to have material adsorbed both on the outer membrane surface and also throughout the membrane.

Magnetic semipermeable aqueous polyethyleneimine microcapsules for monitoring N-nitrosating species in the gastrointestinal tract.

Magnetic polyethyleneimine (PEI) microcapsules have been developed for trapping electrophilic intermediates in the gastrointestinal (GI) tract. The N-nitrosation of these microcapsules at acid pH was found to be linearly dependent on nitrite concentration, without a pH maximum and with efficient conversion to N-nitrosated products which were detected by a total N-nitroso assay procedure. The limit of detection is in the range 1-10 nmol N-nitroso compound, depending on microcapsule preparation conditions. Nuclear magnetic resonance (NMR) indicates that such N-nitrosation is favoured by incomplete protonation of the polyamine. Microcapsules administered orally to rats were recovered magnetically from faeces and showed extensive N-nitrosation when nitrite was administered in the drinking-water.

Oxidative destruction of hydrazines produces N-nitrosamines and other mutagenic species.

As part of the joint International Agency for Research on Cancer-National Cancer Institute program for the evaluation and development of methods for the degradation of chemical carcinogens, four oxidative techniques for the degradation of hydrazines were investigated. The oxidizing agents used were as follows: sodium hypochlorite, calcium hypochlorite, potassium iodate, and potassium permanganate in sulfuric acid. In each case, at least 99% of the hydrazine initially present was destroyed; however, the potential usefulness of these methods was compromised by the formation (in some reaction mixtures) of carcinogenic N-nitroso compounds and/or unknown mutagenic species. Oxidative degradation of hydrazines is recommended only for the decontamination of glassware and for the treatment of spills, for which reductive degradation methods are not suitable.