Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with DeltaNp73.

TP63, a member of the TP53 gene family, encodes two groups of three isoforms (alpha, beta and gamma). The TAp63 isoforms act as transcription factors. The DeltaNp63 isoforms lack the main transcription activation domain and act as dominant-negative inhibitors of transactivation (TA) isoforms. To clarify the role of these isoforms and to better understand their functional overlap with p53, we ectopically expressed each p63 isoform in the p53-null hepatocellular carcinoma cell line Hep3B. All TA isoforms, as well as DeltaNp63alpha, had a half-life of <1 h when transiently expressed and were degraded by the proteasome pathway. The most stable form was DeltaNp63gamma, with a half-life of >8 h. As expected, TA isoforms differed in their transcriptional activities toward genes regulated by p53, TAp63gamma being the most active form. In contrast, DeltaNp63 isoforms were transcriptionally inactive on genes studied and inhibited TA isoforms in a dose-dependent manner. When stably expressed in polyclonal cell populations, TAp63beta and gamma isoforms were undetectable. However, when treated with doxorubicin (DOX), p63 proteins rapidly accumulated in the cells. This stabilization was associated with an increase in phosphorylation. Strikingly, in DOX-treated polyclonal populations, increase in TAp63 levels was accompanied by overexpression of DeltaNp73. This observation suggests complex regulatory cross talks between the different isoforms of the p53 family. In conclusion, p63 exhibits several transcriptional and stress-response properties similar to those of p53, suggesting that p63 activities should be taken into consideration in approaches to improve cancer therapies based on genotoxic agents.

Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-kappaB.

Overexpression of cyclooxygenase-2 (Cox-2) is thought to exert antiapoptotic effects in cancer. Here we show that the tumor suppressor p53 upregulated Cox-2 in esophageal and colon cancer cell lines by inducing the binding of nuclear factor-kappaB (NF-kappaB) to its response element in the COX-2 promoter. Inhibition of NF-kappaB prevented p53 induction of Cox-2 expression. Cooperation between p53 and NF-kappaB was required for activation of COX-2 promoter in response to daunomycin, a DNA-damaging agent. Pharmacological inhibition of Cox-2 enhanced apoptosis in response to daunomycin, in particular in cells containing active p53. In esophageal cancer, there was a correlation between Cox-2 expression and wild-type TP53 in Barrett's esophagus (BE) and in adenocarcinoma, but not in squamous cell carcinoma (P<0.01). These results suggest that p53 and NF-kappaB cooperate in upregulating Cox-2 expression, promoting cell survival in inflammatory precursor lesions such as BE.

Mutagens, N-nitroso compounds and their precursors in gastric juice from patients with and without precancerous lesions of the stomach.

This study examined whether elevated risk of gastric cancer is associated with high levels of total N-nitroso compounds (NOC), their precursors and nitrosation-dependent genotoxins in gastric juice (GJ). An improved method for quantifying total NOC was used and genotoxicity was assayed in E. coli. Results from patients (n = 210) with or without precancerous lesions of the stomach and living in three areas with up to 8-fold variations in gastric cancer risk (U.K., France, Colombia) were compared. The level of nitrite (range < 1-472 mumol/l) was found to increase with the pH of GJ from the three countries and was dependent on country of collection. The levels of NOC (range: < or = 0.01-8.0 mumol/l) in GJ were not affected by stomach histology and country of collection. NOC levels increased linearly with nitrite concentrations, but the slope of the regression line was greater for acidic GJ (pH < or = 4). These data together suggest that chemical nitrosation contributes at least as much as other nitrosation pathways to the intragastric formation of NOC. Acid-catalysed nitrosation of GJ in vitro increased the NOC concentration (range: 7-1332 mumol/l) up to several 1000-fold but this increase was not predictive of gastric cancer risk either by country or by stomach histology. After acid-catalysed nitrosation, direct genotoxicity (SOS-inducing potency) was significantly higher in GJ with original pH > 4 and highest in samples from Colombia. The results (a) provide no support that intragastric total NOC levels are elevated in subjects with precancerous stomach lesions or living in a high risk area for stomach cancer; (b) confirm that a high nitrite level and elevated pH in GJ are strongly associated, the level of nitrite being associated with precancerous stomach conditions only in Colombia; (c) reveal the presence of precursor compounds in GJ, that after nitrosation yield direct mutagens that probably contain NOC and other substances. As their concentrations were significantly higher in achlorhydric subjects and highest in Colombian patients, these data together provide support for a role of intragastrically formed nitrite-derived direct mutagens in gastric cancer aetiology.

Interaction between genetic polymorphism of cytochrome P450-1B1 and environmental pollutants in breast cancer risk.

Cytochrome P450 1B1 (CYP1B1) is implicated in the activation of potentially carcinogenic xenobiotics and oestrogens. The polymorphism of the CYP1B1 gene at codon 432 (Val-->Leu) is associated with change in catalytic function. In a case-series study of breast cancer patients, we investigated the interaction between this polymorphism and environmental exposure. The women carrying the Val CYP1B1 allele and who had lived near to a waste incinerator for more than 10 years had a higher risk of breast cancer than those never exposed with the Leu/Leu genotype (odds ratio of interactions (ORi)=3.26, 95% confidence interval (CI) 1.20-8.84). Also, the Val CYP1B1 allele increased the susceptibility to breast cancer for women exposed during their life to agricultural products used in farming (ORi = 2.18, 95% CI 1.10-4.32). These xenobiotics, mainly organochlorine hydrocarbons, are known to bind to the aromatic hydrocarbon receptor (AhR), and to induce the expression of CYP1B1 enzyme. The excess risk for exposed women with a Val CYP1B1 homo/heterozygous genotype could result from a higher exposure to activated metabolites of pesticides or dioxin-like substances. Also, a higher induction of CYP1B1 enzyme by xenobiotics could increase the formation of genotoxic catechol-oestrogens among exposed women carrying the Val CYP1B1 allele. Our results suggested that the Val CYP1B1 allele increases the susceptibility to breast cancer in women exposed to waste incinerator or agricultural pollutants.

Genetic polymorphism of N-acetyltransferases, glutathione S-transferase M1 and NAD(P)H:quinone oxidoreductase in relation to malignant and benign pancreatic disease risk. The International Pancreatic Disease Study Group.

Carcinogens present in cigarette smoke and diet have been associated with pancreatic cancer. We hypothesized that heterocyclic and aromatic amines implicated in these exposures could be involved as causative agents and that therefore genetic variation in enzymes metabolizing these carcinogens could modify the risk of developing malignant and benign pancreatic disease. The effect of the genetic polymorphism of acetyltransferases (NAT1) and NAT2), glutathione S-transferase M1 (GSTM1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) on the risk of pancreatic diseases (cancer, pancreatitis) was examined in a case-control study. PCR-based assays were used for genotype analysis of genomic DNA from whole blood cells. Samples collected from Caucasian patients with diagnosed pancreatic cancer (n = 81), with non-alcoholic (n = 41) and alcoholic pancreatitis (n = 73) and from asymptomatic control subjects (n = 78) were analysed. The prevalence of GSTM1 null genotype and of NAT2 fast and slow acetylator genotypes and the distribution of frequencies for NQO1 genotypes did not differ in subjects with pancreatic diseases vs controls. For NAT1 slow acetylators a non-significant excess (P = 0.18) was found among pancreatic cancer cases vs controls. There was a significant over-representation of the GSTM1 AB or B genotype in all pancreatic disease cases combined (OR = 2.6; P < 0.05). When concurrent controls were pooled with literature controls (n = 1427), OR was 1.4 (P = 0.08). The results of this study, requiring confirmation, suggest that the polymorphism of GSTM1 and NAT1 enzymes may be associated with a modest increase in susceptibility to pancreatic diseases.

Identification of a second promoter in the human c-ets-2 proto-oncogene.

We localized and characterized a new regulatory element with promoter activity in the human c-ets-2 intron 1. This promoter governs the expression of 5' divergent c-ets-2 transcripts through multiple start sites dispersed within 300 bp. Among the multiple start sites detected, three are major transcriptional initiation points. We detected transcripts initiated from this new promoter in various cell lines such as COLO 320, NBE, or HepG2 cells. This promoter exhibits transcriptional activity when linked to the CAT gene, and deletion constructs reveal that it contains activating and repressing elements. The sequence of the promoter reveals putative binding sites for ETS, MYB, GATA, and Oct factors. In addition, we show that this promoter is functionally conserved in the chicken.

Interrelationships in mice of antipyrine half-life, hepatic monooxygenase activities and liver S9-mediated mutagenicity of aflatoxin B1, benzo[alpha]pyrene 7,8-dihydrodiol, 2-acetylaminofluorene and N-nitrosomorpholine.

To evaluate the predictive value of serum antipyrine half-life AP(T1/2) as an index of hepatic carcinogen metabolism, groups of C57BL/6 and DBA/2 mice were treated with various inducers and inhibitors of cytochrome P-450-dependent monooxygenases (pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), 5,6-benzoflavone (5,6-BF), 3-methylcholanthrene (MC), disulfiram (DIS), 7,8-BF). Groups of mice were also given ethanol (3% in drinking water) for 12 days. Within each group, mean serum AP-(T1/2) was compared with (i) the in vitro activity of hepatic microsomal benzo[alpha]pyrene (BP) 3-hydroxylase, 2-acetylaminofluorene (AAF)-N-hydroxylase and aldrin monooxygenase, and (ii) the liver S9-mediated mutagenicity of aflatoxin B1 (AFB), trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene (BP 7,8-diol), 2-acetylaminofluorene and N-nitrosomorpholine (NMOR) in Salmonella typhimurium strains. Serum AP(T1/2) was only correlated negatively with the activity of BP 3-hydroxylase (P less than 0.001) and aldrin monooxygenase (P less than 0.001). No statistically significant correlation was found between serum AP(T1/2) and liver S9-mediated mutagenicity for any of the four carcinogens. On the basis of these results, we conclude that serum AP(T1/2) may not be a reliable index of the capacity of liver to convert carcinogens into reactive intermediates.

Effect of glutathione and uridine 5'-diphosphoglucuronic acid on mutagenesis by benzo[a]pyrene and aflatoxin B1 in the Salmonella/microsome assay.

In the Salmonella/microsome plate or liquid assay, the addition of glutathione (GSH) and uridine 5'-diphosphoglucuronic acid (UDPGA), both cofactors for GSH-S-transferases or UDPGA-transferases, altered the rat-liver microsome-mediated mutagenesis of benzo[a]pyrene (BP) and aflatoxin B1 (AFB). With either BP or AFB, an increased, unchanged or decreased number of revertant colonies of S. typhimurium was observed, depending on the substrate concentration, the source of rat-liver 9000 X g supernatant (S9), the time of incubation and the type of mutagenicity test (liquid or plate assay). Several factors responsible for quantitative changes in the pattern of BP and AFB metabolites under various assay conditions in vitro, which alter the overall mutagenic activity of the parent compound, are discussed.

Some factors determining the concentration of liver proteins for optimal mutagenicity of chemicals in the Salmonella/microsome assay.

In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher concentration of cytosolic fraction. Studies on the glutathione-dependent reduction of BP mutagenicity in plate assays has shown that, in the presence of liver S9 concentrations greater than that required for optimal mutagenicity, the reduction in mutagenicity was related directly to the concentration of liver S9. Thus, in the Salmonella/microsome assay, when the concentration of rat-liver S9 was increased over and above the amount required for the optimal mutagenicity of BP, the mutagenic metabolites of BP were inactivated (by being trapped with cytosolic nucleophiles and/or by enzymic conjugation with glutathione); this effect increased more rapidly than their rate of formation. The concentration of liver S9 for optimal mutagenicity of test compounds requiring activation catalyzed by mono-oxygenases seems, therefore, to be related to the departure from linearity of the relationship between the rate of formation of mutagenic metabolites and the concentration of liver S9.

Lack of mutagenicity of synthetic pyrethroids in Salmonella typhimurium strains and in V79 Chinese hamster cells.

Seven pyrethroids, i.e., cypermethrin, permethrin, deltamethrin, bioresmethrin, resmethrin, cismethrin and fenvalerate, were not found to be mutagenic in (a) Salmonella typhimurium strains TA100 or TA98 in the presence or absence of a rat liver activation system using the plate incorporation assay and fluctuation tests, or (b) V79 Chinese hamster cells in the presence or absence of hepatocytes.

Antimutagenic dietary phenolics as antigenotoxic substances in urothelium of smokers.

Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of PhIP-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified ABP-dG adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. These substances appear to be dietary phenolics and/or their metabolites because (i) the anti-mutagenic activity of urine extracts (n=18) was linearly related to their content in phenolics; (ii) the concentration ranges of these substances in urine extracts were similar to those of various plant phenols (e.g., quercetin, isorhamnetin) for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; (iii) treatment of urines with beta-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; (iv) urinary extracts inhibited non-competitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Overall, our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells.

Levels of direct-acting mutagens, total N-nitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China.

A high gastric cancer mortality in Fujian province (Peoples Republic of China) has been associated with the consumption of certain salted fermented fish products such as fish sauce (FS). We have investigated the levels and nature of N-nitroso compounds (NOC) and genotoxins present, before and after nitrosation, in 49 FS samples collected from villages in this high-risk area, pooled into six samples. The concentrations of total NOC before nitrosation ranged from 0.2 to 16 mumoles/l, and after nitrosation at pH 2 and pH 7, they rose by up to 4800- and 100-fold, respectively. In nitrosated samples, 40-50% of total NOC was not extractable into organic solvents; volatile N nitrosamines accounted for 1-2% and N-nitrosamino acids for 8-16% of total NOC. None of the FS samples exhibited genotoxic activity, but after nitrosation all were weakly active in the SOS chromotest. The highest SOS-inducing potency was observed with nitrosated ethyl acetate extracts of most samples. The formation of methylating agents was measured by incubation of nitrosated FS with DNA and subsequent analysis of 7-methylguanine adduct. 2 of the 6 nitrosated FS samples caused a slight increase in DNA methylation. 1 pooled home-made FS sample (the only one tested) contained tumour promoter-like substances, as measured by expression of certain EBV genes in Raji cells. HPLC fractionation of ethyl acetate extracts of FS samples allowed identification of three UV-absorbing peaks that, upon nitrosation, produced direct-acting genotoxins. This genotoxicity was partly ascribed to the formation of nitrite-derived arene diazonium cations that were characterized by a coupling reaction with N-ethyl-1-naphthylamine and thin-layer chromatography.

Characterization and identification of 6 mutagens in opium pyrolysates implicated in oesophageal cancer in Iran.

Previous epidemiological studies have indicated an association between the ingestion of opium pyrolysates, dietary deficiencies, and a high incidence of oesophageal cancer in subjects in north-east Iran. Laboratory studies have shown that pyrolysates of opium and particularly of morphine, a major opium alkaloid, are highly mutagenic in bacteria and induce sister-chromatid exchanges in mammalian cells after metabolic activation. We now report the ability of these pyrolysates to transform Syrian hamster embryo cells in culture and present some evidence for their carcinogenicity in mice and hamsters following topical, subcutaneous, intratracheal and intragastric administration. 6 of the most abundant mutagenic compounds present in morphine pyrolysate were isolated and purified by high-performance liquid chromatography and characterized by gas chromatography/mass spectrometry and 1H-Fourier transform nuclear magnetic resonance spectroscopy. These hitherto unknown compounds, all containing a hydroxy-phenanthrene moiety, were identified as: 3-methyl-3H-naphth[1,2-e]indol-10-ol; 1,2-dihydro-3-methyl-3H-naphth[1,2-e]indol-10-ol; 6-methylaminophenanthren-3-ol; 2-methylphenanthro[3,4-d] [1,3]oxazol-10-ol; 2,3-dimethyl-3H-phenanthro[3,4-d]imidazol-10-ol and 2-methyl-3H-phenanthro[3,4-d]imidazol-10-ol. Mutagenicity in Salmonella typhimurium TA98 of these compounds increased in the order listed, the last compound being 35 times more active than benzo[a]pyrene. The mechanisms, by which these mutagens are formed and metabolically activated are discussed.

Identification, occurrence and mutagenicity in Salmonella typhimurium of two synthetic nitroarenes, musk ambrette and musk xylene, in Indian chewing tobacco and betel quid.

During N-nitrosamine analysis of extracts of betel quid with tobacco and of the saliva of chewers of betel quid with tobacco for N-nitrosamines using a Thermal Energy Analyzer, two unknown compounds were detected. They were identified as synthetic nitro musks, musk ambrette (5-tert-butyl-1,3-dinitro-4-methoxy-2-methylbenzene, CAS No. 83-66-9) and musk xylene, (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene, CAS No. 81-15-2), by gas chromatography-mass spectrometry and Fourier transform nuclear magnetic resonance spectroscopy. These compounds were detected in several samples of betel quid with tobacco and in perfumed tobacco used for chewing in India in amounts ranging from 0.45-23.5 mg/g wet weight. Musk ambrette was found to be mutagenic in Salmonella typhimurium TA100 requiring metabolic activation by rat-liver postmitochondrial supernatant but musk xylene lacked mutagenicity.

Synthesis, structure-activity relationships and a reaction mechanism for mutagenic N-nitroso derivatives of glycosylamines and Amadori compounds--model substances for N-nitrosated early Maillard reaction products.

A series of nine glycosylamines and an Amadori compound were synthesized, together with their N-nitroso derivatives. Their structures were established by physico-chemical and spectroscopic data and elemental analyses. The N-nitroso compounds were further characterized by denitrosation with hydrogen bromide-acetic acid, followed by detection of the liberated NO by a chemiluminescence detector. N-Nitroso derivatives of N-p-nitrophenyl/p-methylphenyl/p-carboxyphenyl pentopyranosylamines, N-p-methylphenyl-1-deoxy-D-fructosylamine (the Amadori compound) and N-3-ethylindole-D-xylopyranosylamine were shown to be direct-acting mutagens in Salmonella typhimurium TA100. The activity of some of the compounds was similar to that of N-ethyl-N-nitrosourea. Their mutagenic activity was shown to depend on the structure of the amine and the sugar moieties and to require the presence of free hydroxyl groups in the sugar. The mutagenicity of N-nitrosoglycosylamines was attributed to their hydrolysis to arenediazonium cations. The formation of these compounds was detected by azo-coupling with N-ethyl-1-naphthylamine, using spectrophotometric and mass spectrometric analyses. These data implicate arene(alkyl)diazonium cations as the ultimate mutagens of N-nitrosoglycosylamines (and possibly of N-nitroso Amadori compounds), a little-explored class of N-nitroso compounds that may be formed in vivo.

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.

p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter.

The tumor suppressor p53 protein is activated by genotoxic stress and regulates genes involved in senescence, apoptosis and cell-cycle arrest. Nine p53 isoforms have been described that may modulate suppressive functions of the canonical p53 protein. Among them, Delta133p53 lacks the 132 proximal residues and has been shown to modulate p53-induced apoptosis and cell-cycle arrest. Delta133p53 is expressed from a specific mRNA, p53I4, driven by an alternative promoter P2 located between intron 1 and exon 5 of TP53 gene. Here, we report that the P2 promoter is regulated in a p53-dependent manner. Delta133p53 expression is increased in response to DNA damage by doxorubicin in p53 wild-type cell lines, but not in p53-mutated cells. Chromatin immunoprecipitation and luciferase assays using P2 promoter deletion constructs indicate that p53 binds functional response elements located within the P2 promoter. We also show that Delta133p53 does not bind specifically to p53 consensus DNA sequence in vitro, but competes with wild-type p53 in specific DNA-binding assays. Finally, we report that Delta133p53 counteracts p53-dependent growth suppression in clonogenic assays. These observations indicate that Delta133p53 is a novel target of p53 that may participate in a negative feedback loop controlling p53 function.

Synthesis, analysis and mutagenic activity of N-nitroso derivatives of glycosylamines and Amadori compounds: nitrosated model substances for the early Maillard reaction products.

A series of nine glycosylamines and an Amadori compound and their N-nitroso derivatives were synthesized. The structures were ascertained by spectroscopy and elemental analysis. The N-nitroso compounds were further characterized by denitrosation with hydrogen bromide-acetic acid, followed by detection of the liberated NO by a chemiluminescence detector. N-Nitroso derivatives of N-p-nitrophenyl/p-methylphenyl/p-carboxyphenyl pentosylamines, N-p-methylphenyl-1-deoxy-D-fructosylamine (Amadori compound) and N-3-ethylindole-D-xylosylamine were shown to be directly-acting mutagens in Salmonella typhimurium TA100. The activity of some of the compounds was similar to that of N-ethyl-N-nitrosourea. Their mutagenic activity was shown to be dependent on the structure of the amine and the sugar moieties and requires the presence of free hydroxyl groups in the sugar. The mutagenicity of N-nitrosoglycosylamines was attributed to their hydrolysis to arene diazonium cations. Their formation was detected via azo-coupling with N-ethyl-1-naphthylamine, using spectrophotometric and mass-spectrometric analyses. Our data implicate arene (alkyl) diazonium cations as the ultimate mutagens of N-nitrosoglycosylamines and N-nitroso Amadori compounds, a little explored class of N-nitroso compounds which may be formed in vivo.

Substituted hydroxyphenanthrenes in opium pyrolysates implicated in oesophageal cancer in Iran: structures and in vitro metabolic activation of a novel class of mutagens.

Previous epidemiological and laboratory studies have indicated an association between the ingestion of opium pyrolysates, dietary deficiencies and the high incidence of oesophageal cancer in subjects in north-east Iran. Pyrolysates of opium, and particularly of morphine, a major opium alkaloid, were both shown to contain similar highly mutagenic substances that were also clastogenic in mammalian cells and which transformed hamster embryo cells in culture. We now report the isolation and characterization of nine of the most abundant mutagenic compounds present in morphine pyrolysates, using h.p.l.c, GC-MS and n.m.r. spectroscopy. The hitherto unknown compounds, all containing a hydroxyphenanthrene moiety, were identified as: I, 3-methyl-3H-naphth[1,2-e]indol-10-ol; II, 1,2-dihydro-3-methyl-3H-naphth[1,2-e]indol-10-ol; III, 1-methyl-1H-naphth[2,1-g]indol-10-ol; IV, 2-methylphenanthro[3,4-d]-[1,3]oxazol-10-ol; V, 6-methylaminophenanthren-3-ol; VI, 2-methyl-3H-phenanthro[3,4-d]imidazol-10-ol; VII, 1,2-dimethyl-1H-phenanthro[3,4-d]imidazol-10-ol; VIII, 2,5-dimethyl-3H-phenanthro[3,4-d]imidazol-10-ol; and IX, 2-ethyl-3H-phenanthro[3,4-d]imidazol-10-ol. Structures for the heterocyclic rings of compounds IV and VI to IX are tentative. Mutagenicity in Salmonella typhimurium TA98 in the presence of rat liver homogenates increased in the order listed and ranged over four orders of magnitude, IX being 1000 times more active than benzo[a]pyrene. Compounds I and VII were converted by rat liver 9000 g supernatant into phenols and dihydrodiols, implicating arene oxides as ultimate mutagens. The formation and reaction of these arene oxides was shown by trapping experiments in vitro with ethanethiol and subsequent characterization of the ethyl sulfide reaction products. The order of biological activity of compounds I-IX, dependent on the structure of the heterocyclic ring, suggests that carbocations, resonance-stabilized as quinone methides, are their ultimate reactive metabolites. Our results lend additional support to the role of opium pyrolysates as an etiological factor in oesophageal cancer in north-east Iran.