I.K. O'Neill

Apolipoprotein E and methylenetetrahydrofolate reductase genetic polymorphisms in relation to other risk factors for cardiovascular disease in UK Caucasians and Black South Africans

Genetic polymorphisms for apolipoprotein E (apo E) and methylenetetrahydrofolate reductase (MTHFR) are believed to modulate risk of coronary heart disease (CHD) acting through regulation of lipid and homocysteine metabolism, respectively. The distributions of apo E and MTHFR alleles in Black South Africans, a population with a low CHD incidence, and UK Caucasians from the Cambridge area, with a higher CHD incidence, were therefore compared. Clinically healthy volunteers (207), including 107 UK Caucasians from the Cambridge area and 100 Black South Africans, participated in the study. Apo E and MTHFR genotypes were determined in all of them. Analyses for serum total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and plasma fibrinogen were carried out in 65 UK Caucasians and 60 Black South Africans. The apo E epsilon4 allele, which is associated with elevated CHD risk, was present in 48% of Black South Africans compared to 20.8% of Caucasians (P < 0.0001); however, both total and LDL cholesterol levels in Black South Africans were 18-32% lower than in Caucasians with similar apo E genotypes. Hyperhomocysteinemia-causing MTHFR 677T variant was detected in only 20% of Black South Africans (no homozygotes) versus 56% of Caucasians with 12% homozygotes (P<0.0001). Our findings suggest that the potentially unfavourable pattern of apo E allele distribution in Black South Africans does not result in increased CHD incidence due to protection by dietary and/or other life style related factors. The exceptionally low frequency of MTHFR mutant homozygotes in this population suggests that this polymorphism should not be regarded as an important CHD risk factor among Black South Africans.

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.

Conversion of IQ to 7-OHIQ by gut microflora.

The rates of conversion of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) to its reportedly mutagenic 7-keto derivative (7-OHIQ) by intestinal bacteria from humans, mice, and rats were compared. IQ was metabolized faster by cecal contents from rats or mice than by human fecal samples (113 and 87 mumol 7-OHIQ formed/hr/g cecal contents, respectively, vs. 12.3 mumol/hr/g feces). Cecal contents from germ-free rats colonized with human fecal bacteria [human flora-associated (HFA) rats] converted IQ to 7-OHIQ at rates generally lower than contents from rats colonized with their native flora. Diet had a marked effect on IQ metabolism by HFA rat cecal contents. The rate of IQ conversion to 7-OHIQ was increased in rats fed a diet high in beef dripping compared with that in rats fed a low-fat control diet. A diet high in olive oil, however, did not produce an increase in the IQ conversion rate. Addition of fiber to a purified diet increased the rate of IQ metabolism in the following order: sugar beet fiber > wheat bran > oat bran fiber > fiber-free diet. In a further study, HFA rats were fed human diets altered independently in their fat, fiber (wheat bran), and beef contents. The high-fiber diet produced the greatest increase in IQ conversion rate, followed by the high-fat diet. The diet with a high beef content and the control diet (low levels of all 3 macrocomponents) produced similarly low rates of IQ conversion. Material from incubations of IQ with HFA rat cecal contents, assumed to be 7-OHIQ on the basis of chromatographic behavior, was confirmed to be directly mutagenic, producing approximately 800 His+ revertants per microgram with S. typhimurium TA98.

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

Abstract 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 1 H-Fourier transform nuclear magnetic resonance spectroscopy. These hitherto unknown compounds, all containing a hydroxy-phenanthrene moiety, were identified as: 3-methyl-3 H -naphth[1,2- e ]indol-10-ol; 1,2-dihydro-3-methyl-3 H -naphth[1,2- e ]indol-10-ol; 6-methylaminophenanthren-3-ol; 2-methylphenanthro[3,4- d ][1,3]oxazol-10-ol; 2,3-dimethyl-3 H -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.

Occurrence in human urine of new sulphur-containing N-nitrosamino acids N-nitrosothiazolidine 4-carboxylic acid and its 2-methyl derivative, and their formation

SummaryTo quantitate endogenous nitrosation reactions in man, the quantity of N-nitrosoproline (NPRO) excreted in the urine after ingestion of proline and/or nitrate was estimated. When this monitoring method (NPRO test) was applied in clinical and field studies, several hitherto unidentified N-nitroso compounds were frequently detected. These were recently identified as sulphur-containing N-nitrosamino acids, N-nitrosothiazolidine 4-carboxylic acid (NTCA), and trans- and cis-isomers of N-nitroso-2-methylthiazolidine 4-carboxylic acid (NMTCA).NTCA and NMTCA were readily formed in vitro following nitrosation at acidic pH of the respective precursor, thiazolidine 4-carboxylic acid (TCA) or of 2-methylthiazolidine 4-carboxylic acid (MTCA). As the latter compounds can be formed by reaction of l-cysteine with formaldehyde or acetaldehyde, respectively, NTCA and NMTCA were also formed by reacting l-cysteine with the respective aldehyde and with nitrite at optimal pH (2.5 for NTCA and 4.5 for NMTCA).Up to 95% of NTCA and NMTCA given orally to fasted rats was recovered as such in urine and faeces within 2 days. Administration of TCA or MTCA, together with nitrite increased the urinary excretion of NTCA and NMTCA, as did co-administration of l-cysteine, nitrite, and the respective aldehyde.NTCA and NMTCA were also detected in the 24-h urine of human volunteers, and smokers tended to excrete higher levels than nonsmokers. Daily excretion levels varied, however, and a diet supplemented with ascorbic acid significantly descreased the total amount of nitrosamino acids. NTCA and NMTCA may occur in human urine as a result of (i) intake of preformed N-nitroso compounds; (ii) intake of thiazolidine 4-carboxylic acid or its 2-methyl derivative and subsequent nitrisation in vivo; (iii) endogenous two-step synthesis by the reaction of l-cysteine with the respective aldehyde and a nitrosating agent.Thus, measurement of NTCA and NMTCA together with NPRO in urine may provide an index for the exposure of human subjects to nitrosamines or their precursors, i. e., nitrosating agents, certain aldehydes, or aldehyde-generating compounds. Our data demonstrate unequivocally that N-nitroso compounds are formed in the human body, as suggested previously by Druckrey. Their relevance to human cancer at specific sites should now be investigated.

Presence in human urine of a new N-nitroso compound, N-nitrosothiazolidine 4-carboxylic acid

Urine samples collected in several countries from human subjects showed the presence of a number of N-nitroso compounds not previously identified. By several separative procedures and by comparison with authentic material, the major unknown N-nitroso compound was shown to be N-nitrosothiazolidine 4-carboxylic acid (NTCA). Although its origin in human urine is unknown, thiazolidine 4-carboxylic acid, the easily nitrosatable amine precursor, can be formed by reaction of formaldehyde with cysteine in vivo and in vitro. Thus measuring NTCA excreted in the urine may allow monitoring exposure of human subjects to precursors like formaldehyde and NO-3/NO-2.

Magnetic polyethyleneimine (PEI) microcapsules as retrievable traps for carcinogen electrophiles formed in the gastrointestinal tract

Semi-permeable magnetic microcapsules containing polyethyleneimine (PEI) have been developed as retrievable carcinogen traps. In vitro, the soluble core PEI and membrane both bound reactive substances of limited aqueous stability, such as from [14C]N-methyl-N-nitrosourea ([14C]NMU), and aqueous stable dyes of molecular weight up to 1000. The core/membrane location ratio of binding was dependent upon membrane characteristics of the microcapsule batch used. Microcapsules administered intragastrically to rats bound up to 0.006% of [14C]dimethylhydrazine ([14C]DMH) and 1.4% of [14C]NMU administered i.p. or intrarectally, respectively. Time-dependency of [14C]DMH binding was consistent with labelling of microcapsules within the small intestine. There were no detectable metabolites from [14C]DMH trapped within the colon, whereas binding of [14C]NMU indicated that microcapsules could bind transient species present within the colon in competition with the faecal bulk. These results indicate that this approach could be used to detect highly unstable and possibly genotoxic substances in situ, hitherto unknown, formed within the intestinal lumen.

Nucleophilic selectivity and reaction kinetics of chloroethylene oxide assessed by the 4-(p-nitrobenzyl)pyridine assay and proton nuclear magnetic resonance spectroscopy

The nucleophilic selectivity (Swain-Scotts constant s) of chloroethylene oxide (CEO), an ultimate carcinogenic metabolite of vinyl chloride, was determined to be 0.71 using the 4-(p-nitrobenzyl)pyridine (NBP) assay (Spears method). The molar extinction coefficient of the adduct formed between NBP and CEO was measured; and the second-order rate constants for the reactions of CEO with NBP and with thiosulfate were estimated at three temperatures. The disappearance of CEO and the formation of chloroacetaldehyde (CAA) and glycolaldehyde (GCA) were followed in D2O or a mixture of D2O/hexadeuterated acetone (acetone-d6), using Fourier transform proton nuclear magnetic resonance spectroscopy (1H-FTNMR). Evidence was obtained that CEO reacts with chloride ions to yield CAA at a rate constant of about 17 M-1 h-1 in D2O/acetone-d6 (1 : 1, v/v) at 280 K. Under the same conditions, the first-order rate constant kr for the thermal rearrangement of CEO into CAA was estimated to be approximately 0.41 h-1. These data suggest that the isomerization of CEO may be a minor reaction in physiological saline. These chemical properties of CEO are discussed in relation to the mechanism of vinyl chloride-induced carcinogenesis.

Gastrointestinal monitoring of DNA-damaging agents with magnetic microcapsules.

Semi-permeable, magnetically recoverable, reactive microcapsules of several types were developed for gastrointestinal (GI) monitoring of several kinds of DNA-damaging agents in relation to (i) systematic dietary variations designed to discriminate the GI effects of food components known to modulate colorectal cancer risk, and (ii) then thereby to achieve the identification of a range of endogenous agents and their dietary sources. These microcapsules contained as targets either amino functions (for alkylating agents), 14CH3 functions (to detect cross-linking agents and reactive oxygen species precursors), or a copper porphyrin (for carcinogens having planar molecular structure). Other microcapsules had a cleavable target based on guanine, which is shown to trap endogenous agents and a [14C]BaP metabolites in male F344 rats consuming a putative high-risk diet (high fat, high meat, low fibre non-starch polysaccharide (NSP)), but not significantly when consuming the contrasting low-risk diet. Detailed investigations of the action of fibre NSP and fat showed that increased intake from low to high levels of the British diet range enhanced or decreased several carcinogenesis-relevant end-points more than two-fold. Detection of these disproportionately large effects on microcapsule trapping, hepatic DNA adducts from endogenous agents, colorectal mucosal cell mitoses/micronuclei, endogenous cross-linking agents, and gut microfloral enzyme activities (a) are consistent with epidemiological data on the importance of these components and (b) provide the basis for establishing with microcapsules some potential dietary preventive measures in volunteers.

IARC approaches to monitoring exposure of passive smoking

Existing information on the nature of passive smoking shows many pitfalls in ascertaining carcinogenic exposures. Two current activities of the International Agency for Research on Cancer are described: presentation of standardized monitoring methods for passive smoking; and an international study to explore biochemical measurements in relation to passive smoking history as assessed by interview.