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N J Gooderham

Publications and source records attributed to N J Gooderham.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mutational spectra of the dietary carcinogen 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine(PhIP) at the Chinese hamsters hprt locus.

The mutagenic 'fingerprint' of the cooked food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined in a Chinese hamster cell line genetically engineered to express human CYP1A2 (XEMh1A2-MZ). The parental Chinese hamster V79 and XEMh1A2-MZ cells were exposed to PhIP at various concentrations for 24h. There was a dose-dependent increase in frequency of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus only in the metabolically competent XEMh1A2-MZ cells. The mutant frequency ranged from 25 to 90 X 10(-6) with final concentrations of 2.5 to 100 microM PhIP compared to 8 X 10(-6) in the solvent controls and the V79MZ cells. The molecular nature of the PhIP-induced mutations in XEMh1A2-MZ cells was determined by examining DNA sequence modifications at the hprt locus in forty five 6-thioguanine resistant (6-TGr) mutant clones. Single base substitutions predominantly GC-->TA transversions, were the major class of PhIP-induced mutation. However, a -1 frameshift 'hotspot' in a 5'-GGGA sequence was also observed. With the exception of a compound modification, all of the PhIP-induced mutations involved G.C base pairs. This is consistent with the previously observed PhIP-induced mutations in cultured mammalian cells and 32P-postlabelling experiments that show PhIP adducts to the guanine base and that major adduct is at the C8 position. Furthermore, nearly all of these mutations involved guanine bases on the non-transcribed strand which is possibly indicative of preferential repair of PhIP adducts from the transcribed strand. Nearest neighbor analysis of induced base substitutions indicates a preference for 5' guanine and 3' adenine. These data effectively define a mutation 'fingerprint' for PhIP, which may provide the basis for definitive studies on the role of PhIP in diet associated cancers such as tumours of colon. It is, therefore, intriguing that in their recent report of mutation in tumours of the colon induced by PhIP in male rate Kakiuchi et al. (Proc. Natl Acad. Sci. USA, 92, 910-914) report that four out of eight tumors had identical mutation of the tumour suppressor gene apc which is comprised of a -1 G frameshift in a 5'-GGGA sequence.

Animals↗
PubMed1996

Organ distinctive mutagenicity in MutaMouse after short-term exposure to PhIP.

We have investigated PhIP-induced mutagenicity in various tissues (kidney, liver, large and small intestine) using a transgenic mouse model (MutaMouse). In addition to tissue specific mutagenesis, we measured the binding of [14C]PhIP to MutaMouse mice blood proteins (haemoglobin and albumin), to obtain a quantitative estimate of carcinogen exposure and activation and their relationship to mutagenesis. Short-term (4 days) treatment of MutaMouse mice with [14C]PhIP by oral gavage resulted in the dose-dependent accumulation of radiolabelled material bound to haemoglobin and serum albumin. PhIP, at the highest dose (20 mg/kg), caused a 5.9-fold increase in the mutation frequency in the large intestine, a 4.2-fold increase in the mutation frequency in the small intestine but only a marginal 1.6-fold increase in the liver. However, there was no significant increase in mutations in the kidney at this dose. In contrast, there were no significant differences in any of these tissues between the vehicle control and the two lower doses (2.0 and 0.2 mg/kg respectively). These results are discussed in relationship to those previously reported for PhIP at the Dlb-1 locus.

Administration, Oral↗
PubMed1995

Extra-nuclear location of histones in activated human peripheral blood lymphocytes and cultured T-cells.

Dextrin-2-sulphate (D2S) is a sulphated polysaccharide which inhibits human immunodeficiency virus type 1 infection of T-cells by binding to the cell surface. During our investigations of the nature of this interaction, a cell membrane fraction was prepared by ultracentrifugation from the T-cell line, HPB-ALL. Separation of membrane proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and analysis for binding proteins using ligand blotting showed that 3H-D2S bound, in a saturable and displaceable manner, to two regions corresponding to molecular weights of 14,000-18,000 and 28,000-32,000. The N-terminal sequences of two of the major protein components in the 14,000-18,000 region were consistent with those of histones H2B and H3. The presence of histone H2B in the cell membrane preparation was confirmed by immunoblotting and enzyme-linked immunosorbent assay using a specific antibody. Histone standards were used to determine the level of each histone in the cell membrane fraction. In addition, the binding of 3H-D2S to purified histone standards was quantified. These results show that all of the binding of 3H-D2S to proteins in the 14,000-18,000 region of the cell membrane preparation can be attributed to the histones present. In contrast to HPB-ALL cells, a cell membrane fraction from freshly isolated human peripheral blood lymphocytes contained very low levels of histones. However, after culture with phytohaemagglutinin for 3 days the cell membrane fraction contained greatly increased levels of histones. To exclude the possibility of contamination of the cell membrane preparation with histones derived from the nucleus, cell membranes were also prepared using an affinity-based method using polyethyleneimine-cellulose. Immunoblotting of adsorbed plasma membranes showed the presence of histone H2B. SDS-polyacrylamide gels stained for protein also indicated that the preparation contained histones H1, H2A, H3 and H4. In further experiments whole cells were used to avoid contamination from nuclear proteins. Lactoperoxidase mediated 125I labelling, a method specific for radiolabelling cell surface proteins, confirmed the presence of histones H2B, H3 and H4 on the surface of HPB-ALL cells. Also, incubation of HPB-ALL cells or phytohaemagglutinin-activated peripheral blood lymphocytes with D2S caused displacement of histones from the cell surface into the supernatant without altering cell viability. In addition, immunocytochemistry of freshly isolated peripheral blood lymphocytes showed that histone H2B was located predominantly in the nucleus. However, in phytohaemagglutinin-activated peripheral blood lymphocytes immunoreactive material was also prominent in the endoplasmic reticulum and on the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Enzymatic studies of the activation of heterocyclic food mutagens in man.

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are amongst the most abundant of the heterocyclic aromatic amines formed during the cooking of beef. Both compounds are genotoxic and carcinogenic in rodents. These effects are manifested only after activation of the amines by P450. Human liver is very active at converting these amines to mutagenic products. Studies in vitro have established that, for both amines, mutagenicity with human liver microsomes is entirely via the N-hydroxylamine, essentially the only oxidation product of either amine. Both N-hydroxylation and mutagenicity of the amines can be almost completely inhibited by furafylline, a potent and highly selective inhibitor of CYP1A2 in man. These data, together with the work of others, show that the N-hydroxylation and hence the mutagenicity of both MeIQx and PhIP in man is catalyzed almost exclusively by CYP1A2. Liver from cynomolgus monkeys, unlike that from humans and marmosets, is very poor at activating MeIQx or PhIP to mutagenic products. Studies with anti-peptide antibodies of defined specificity revealed that this is due to the absence of CYP1A2, suggesting that this species should not be used to assess the possible risk posed to man by these amines. The marmoset would be a better species for this purpose.

Animals↗
PubMed1995

Systemic exposure to dietary heterocyclic amines in man.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-I-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed during the cooking of meat and account for a significant proportion of the mutagenic material present in cooked beef. The amines are not directly mutagenic but are converted to active intermediates by P450. Studies in vitro with human liver have shown that N-hydroxylation catalyzed by CYP1A2 is the major pathway of oxidation of MeQx and PhIP and is solely responsible for the generation of mutagenic species. In the studies reported in this paper it is demonstrated that both MelIQx and PhIP are well absorbed and extensively metabolized following ingestion of amine-containing beef by humans. Experiments with furafylline, a potent and selective inhibitor of human CYP1A2, reveal that more than 90% of MeIQx and 70% of PhIP are N-hydroxylated in vivo, probably presystemically in the liver.

Animals↗
PubMed1994

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a potent mutagen in the mouse small intestine.

Mutations in long lived stem cells are critical events in carcinogenesis. The Dlb-1 assay detects intestinal stem cell mutation at the Dlb-1 locus in Dlb-1a/b heterozygous mice by visualizing mutated clones of epithelial cells in situ which do not bind the lectin Dolichos biflorus agglutinin. We have used this assay to show that the food-derived heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent intestinal mutagen when administered either i.p. or p.o. This contrasts with the inactivity of the structurally related mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the assay which we have described previously. Immunocytochemical localization of the P-450 enzyme CYP1A2, which is responsible for the primary activation of these mutagens, shows that in untreated mice it is present in liver hepatocytes and in occasional villus epithelial cells but is absent from the target intestinal stem cell population. In addition, liver microsomes, unlike intestinal microsomes, were able to convert PhIP to the proximate mutagen N-hydroxy-PhIP. CYP1A2 immunoreactivity in beta-napthoflavone-induced animals was elevated in liver hepatocytes and increased to a lesser extent in duodenal villus epithelial cells. Treatment with beta-napthoflavone produced an unexpected 46% decrease in the number of Dlb-1 mutations in response to PhIP. Following treatment with PhIP, there was no difference in the number of Dlb-1 locus mutations between the proximal and distal ends of the small intestine in uninduced animals, indicating that the bile duct is unlikely to be responsible for transport of mutation inducing metabolites of PhIP to the small intestine. Our results demonstrate that metabolic activation of an indirect acting genotoxic agent can occur at a site other than the target tissue, and absence of the enzymes required for activation of a mutagen does not necessarily protect that tissue from its genotoxic effects.

Animals↗
PubMed1994

CYP1A2-catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans.

The contribution of CYP1A2 to the metabolism of the dietary heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in vivo in humans, has been determined with furafylline, a highly selective inhibitor of this enzyme. The inhibitory potential of furafylline in vivo was first assessed by determining its effect on clearance of phenacetin to paracetamol by the model CYP1A2-dependent O-deethylation pathway. Furafylline inhibited this reaction by > 99% in all subjects, thus demonstrating its applicability to determining the contribution of CYP1A2 to a given reaction in vivo. A group of 6 healthy male volunteers received either placebo or 125 mg furafylline, in a double-blind balanced crossover design, 2 h prior to consuming a test meal of fried beef containing a known amount of amines. The excretion of PhIP and MeIQx in urine was determined during the subsequent 28 h, using gas chromatography-mass spectrometry. Following furafylline, the excretion of unchanged MeIQx increased 14.3-fold, while that of PhIP increased 4.1-fold (P < 0.01, paired t test). Elimination of both amines was first order and very rapid, with half-lives of < 5 h. The elimination rate constants did not change following furafylline, suggesting that total clearance is limited by hepatic blood flow. Because the elimination of the amines was first order, it was possible to calculate the contribution of CYP1A2 to the clearance of the amines. CYP1A2-catalyzed metabolism accounts for 91% of the elimination of ingested MeIQx and 70% of ingested PhIP, most likely via N-hydroxylation.

Adult↗
PubMed1994

Metabolism of the food derived mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) by human liver microsomes.

Animal studies have shown that 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) undergoes both activation to a genotoxic metabolite and detoxication, catalysed by CYP enzymes. In this study, using direct chemical analysis, we have examined PhIP metabolism by the microsomal fraction of human liver for comparison to that occurring in animals. PhIP was incubated with human liver microsomes in the presence of an NADPH regenerating system and the reaction mixture then analyzed by HPLC. Only one metabolite, identified as N-hydroxy PhIP, was produced. The N-hydroxylation of PhIP by human liver microsomal fraction obeyed Michaelis-Menten kinetics, with a Km of 55 microM and a Vmax of 666 pmol/min/mg protein. Furafylline, a potent and specific inhibitor of CYP1A2 in man, inhibited this reaction by > 95%, with an IC50 of 0.6 microM. PhIP inhibited high affinity phenacetin O-deethylase activity of human liver microsomes, an activity catalysed specifically by CYP1A2, with an IC50 of about 80 microM. These data indicate that, in human liver microsomes, N-hydroxylation is the only route of oxidative metabolism of PhIP, yielding a genotoxic species, and that this reaction is catalysed almost exclusively by CYP1A2. Furthermore, the exclusive oxidative activation of PhIP by human liver is in direct contrast to PhIP metabolism in rodents and non-human primates where oxidative detoxication products predominate.

Carcinogens↗
PubMed1994

Infection by HIV-1 blocked by binding of dextrin 2-sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T-cells.

1. Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV-1. Using the T-cell lines, C8166 and HPB-ALL, and the laboratory adapted strains of HIV-1.MN, HIV-1.IIIb and HIV-1.RF, dextrin 2-sulphate (D2S) combined the best combination of high anti-HIV-1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity. It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230-3700 nM depending upon the primary viral isolate tested. 2. In saturation binding studies, [3H]-D2S bound to a cell surface protein on HPB-ALL cells in a specific and saturable manner with a Kd of 82 +/- 14 nM and a Bmax of 4.8 +/- 0.3 pmol/10(6) cells. It bound to other human T-cell lines in a similar manner. 3. There was very little binding of [3H]-D2S to freshly isolated PBMN cells (Bmax 0.18 +/- 0.03 pmol/10(6) cells) and these cells could not be infected by HIV-1. Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL-2 did not significantly change the Bmax of [3H]-D2S. In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 micrograms ml-1) for 72 h had a Bmax of [3H]-D2S binding of 7.2 +/- 0.1 pmol/10(6) cells and these cells could be infected by HIV-1. Removal of the PHA and further culture of the PBMN cells in LGM containing IL-2 resulted in a fall in the Bmax to 2.0 +/- 0.1 pmol/10(6) cells. The Kd of binding did not change significantly during the course of these experiments.4. [3H]-D2S did not bind to freshly isolated erythrocytes or to erythrocytes which had been cultured in PHA for 72 h.5. These results suggest that there is a relationship between the expression of the [3H]-D2S binding protein on the plasma membrane of PBMN cells and the susceptibility of these cells to infection by HIV- 1.

Anticoagulants↗
PubMed1993

Activation of the food derived carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by rat pleural cavity inflammatory cells.

There is an increased risk of developing neoplastic disease at sites of chronic inflammation. We have used a model of rat pleural cavity inflammation induced with carrageenan to obtain inflammatory cells comprising predominantly phagocytes (macrophages, monocytes and neutrophils) to examine their ability to activate chemical carcinogens. Treatment of these cells with phorbol-12-myristate-13-acetate (PMA, 15 mM) to stimulate respiratory burst, resulted in a rapid release of reactive oxygen species. Incubation of the cooked food promutagen 2-amino-3,8-dimethylimadazo-[4,5-f]quinoxaline (MeIQx) with PMA (or phorbol dibutyrate or opsonised zymosan or silica) stimulated pleural cavity phagocytes, generated highly electrophilic products which were mutagenic in an Ames Salmonella mutagenicity assay. whereas resting cells (no PMA) had negligible activity. Mutagenic activation of MeIQx by PMA stimulated cells could be reduced by inhibitors of active oxygen such as mannitol, benzoate, dimethyl sulphoxide, superoxide dismutase and catalase and by inhibition of myeloperoxidase activity. In contrast the very potent, broad spectrum cytochrome P450 inhibitor 8-methoxypsoralen had no effect, suggesting the reaction was not cytochrome P450 dependent. Activation of MeIQx by PMA stimulated cells could be abolished by the protein kinase C inhibitor staurosporin, confirming the importance of protein kinase C in the reaction. Furthermore, performing incubations in the presence of fluoride (10 mM) to directly stimulate adenylyl cyclase avoided the requirement for phorbol ester in the activation process. These data show that phagocytes stimulated to release active oxygen species can activate MeIQx to a mutagenic derivative. Mutagenic activation of MeIQx in this system was dependent upon the generation of active oxygen via signal transduction pathways involving protein kinase C and adenylyl cyclase. We suggest that the mechanism of MeIQx activation by phagocytes probably involves one electron oxidation mediated by active oxygen species.

Animals↗
PubMed1993

Molecular dosimetry of the food-borne carcinogen MeIQx using adducts of serum albumin.

Incubation of mouse serum albumin with the food borne carcinogen [2-14C]-Amino-3,8,-dimethylimidazo[4,5-f]quinoxaline (14C-MeIQx) in the presence of mouse hepatic microsomes and an NADPH-regenerating system in vitro resulted in the formation of adducts of MeIQx with albumin, which increased proportionately with time for at least 120 min (approximately 1 pmol equivalents/mg of protein/min). We have previously shown in male Swiss Webster mice in vivo that 14C-MeIQx bound covalently to serum proteins and that the formation of adducts was dose dependent. 14C-MeIQx (100 mg/kg, i.p.) was administered to male (MF1) mice which were killed 24 h later. Serum albumin was purified by affinity chromatography and covalent binding of 14C-MeIQx was assessed. Total covalent binding of MeIQx to albumin was 14.0 +/- 5.2 pmol per mg albumin, which was 5-fold greater than to haemoglobin. Following mild acid hydrolysis, 1.25 pmol MeIQx per mg albumin was liberated as free amine, as determined by gas chromatography negative ion mass spectrometry (GC-MS). This represents 9% of total MeIQx adducted to albumin in vivo (cf 1.3% adducted to haemoglobin). These results suggested that adducts of MeIQx with serum albumin should provide a significantly more sensitive dosimeter than those with haemoglobin. We therefore investigated this approach with serum protein samples from three volunteers. Human serum albumin and non-serum albumin protein fractions were separated by affinity chromatography, before being subjected to GC-MS analysis for hydrolysable adducts of MeIQx. The levels of MeIQx in control samples, and from the release of the putative sulphinamide adducts in hydrolysed samples were below the limits of detection of the GC-MS assay (29 +/- 2.6 amol MeIQx/mg albumin). Despite an increase of 2 orders of magnitude in sensitivity, compared with haemoglobin, it is unlikely that the sulphinamide adduct of MeIQx with human serum albumin can be used as a dosimeter for human aminoimidazoazaarene exposure.

Adult↗
PubMed1992

Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef.

During the cooking of beef, the genotoxic heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed. Little is known about the fate of these compounds in humans or the factors affecting it. We have developed assays based on capillary column gas chromatography-negative ion mass spectrometry capable of the simultaneous measurement of MeIQx, DiMeIQx, and PhIP in cooked meat and in human urine using stable isotope labeled analogues. Ten normal, healthy male volunteers were invited to consume a standard cooked meat meal (400-450 g lean beef, cooked as patties on a griddle hotplate) on four separate occasions over a period of 14 months. Following consumption of the test meals, urine was collected from 0 to 8 h, during which time all free amines were excreted and analyzed for MeIQx, DiMeIQx, and PhIP. Subjects ingested 240 +/- 9 (SEM) g cooked meat, which contained 2.2 +/- 0.2 ng MeIQx/g meat, 0.7 +/- 0.1 ng DiMeIQx/g meat, and 16.4 +/- 2.1 ng PhIP/g meat. The variability in relative systemic bioavailability was assessed from the percentage of ingested amine excreted unchanged in the urine. Subjects excreted 2.1 +/- 1.1% of MeIQx and 1.1 +/- 0.5% of PhIP ingested as unchanged amine in the urine. Levels of DiMeIQx in urine, if present, were below the sensitivity of our assay (20 pg/ml) and could not be detected in any of the samples analyzed. Irrespective of dose, urinary excretion of unchanged MeIQx or PhIP (expressed as a percentage of the ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low and the pooled interday (intrasubject) coefficients of variation for both compounds were only 19 and 3.4%, respectively. In contrast, inter-subject (intraday) variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Based on these studies, it should be possible to use the percentage excretion of MeIQx and PhIP to assess the relative bioavailability of these compounds in humans.

Adult↗
PubMed1992

N-hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [13C,15N2]MeIQx and [14C]MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M+H]+) from the parent compound, MeIQx and at 230/233 ([M+H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M+H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.

Carcinogens↗
PubMed1991

The measurement of MeIQx adducts with mouse haemoglobin in vitro and in vivo: implications for human dosimetry.

We have investigated covalent binding of radiolabelled [14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to mouse haemoglobin in vitro and in vivo. Furthermore, we report the development of a capillary column gas chromatography negative ion mass spectrometry (GC-MS) assay capable of detecting MeIQx liberated from haemoglobin after acid or base hydrolysis. Following microsomal activation, the amount of radiolabelled material associated with haemoglobin in vitro increased with incubation time to 0.67 +/- 0.15 nmol/mg haemoglobin at 2 h (initial concentration 0.47 mM [14C]MeIQx, mean +/- SD, n = 6). Hydrolysis of these samples with acid revealed that 47-60% of the radiolabelled material covalently bound to haemoglobin was acid labile. Of this, 7.2-9.8% was recovered as MeIQx as determined by GC-MS. This liberated fraction should reflect the amount of sulphinic acid amide present which is formed when N-hydroxy-MeIQx reacts with sulphydryl-containing amino acids present in haemoglobin. In vivo, no radiolabelled material bound to haemoglobin could be detected in animals treated with the lowest dose of MeIQx (0.2 mg/kg). At higher doses, there was a dose-dependent increase in the covalent binding of radiolabel to haemoglobin (2.0-200 mg/kg). However, the GC-MS assay for hydrolysable adducts of MeIQx yielded detectable quantities of MeIQx (32.2 +/- 17.5 fmol MeIQx/mg haemoglobin) only at the highest dose used. Application of the GC-MS assay to human haemoglobin samples showed that acid-labile adducts of MeIQx, if present, were below the limit of detection of the assay. These results show that levels of sulphinamide adducts of the dietary aromatic amine MeIQx, with haemoglobin, are very low and the implications for future human dosimetry of this carcinogen are discussed.

Animals↗
PubMed1991

Distribution and elimination of [2-14C]amino-3,8-dimethylimidazo [4, 5-f] quinoxaline in mice. Analysis by whole animal autoradiography.

1. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a potent bacterial promutagen and carcinogen, formed when beef is cooked. In mammals, MeIQx is metabolized and activated by liver enzymes, but induces tumours in both hepatic and extrahepatic tissues. 2. Intravenous administration of [14C]MeIQx and whole animal autoradiography has been employed to examine the disposition of MeIQx. Within 10 min, radiolabel was distributed throughout body tissue. Liver levels of radioactivity rapidly rose and remained elevated in comparison to other tissues, throughout the period of study (4 d). 3. At early time points radioactivity accumulated in the stomach, kidney and salivary glands then later in the intestine. Radioactivity was rapidly eliminated from the majority of tissues although did persist in liver and intestines throughout the duration of the study, probably due to covalently bound material. No radioactivity could be detected in the central nervous system, thus neither MeIQx nor its metabolites cross the blood-brain barrier to any significant extent. 4. The rapid elimination of radioactivity from the tissues is in good agreement with the time course of excretion of MeIQx. Irrespective of the route of administration (i.v., i.p., p.o.), a substantial proportion of the labelled material is excreted within 24 h and is present in both urine and faeces in similar quantities. 5. Thus MeIQx is extensively bioavailable, is distributed throughout body tissue and, although the majority is quickly cleared, some remains bound to liver and intestine.

Animals↗