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R C Garner

Publications and source records attributed to R C Garner.

At least 91 records · Page 5Linked to original sources
PubMed1984

Mutagenicity of gastric juice: the importance of controlling histidine concentration when using Salmonella tester strains.

The mutagenic activity of gastric juice has been assessed using bacterial tester strains that undergo reverse mutation (Salmonella typhimurium:his(-)----his(+)). Free histidine, a known source of inaccuracy in this mutation test system, was detected in 42 of 73 juice samples (concentration range 3.5-992.4 micrograms/ml); high histidine concentrations were significantly correlated with hypochlorhydria. The effect of histidine was controlled by using a pre-incubation modification of the Salmonella fluctuation test in which juice samples and their corresponding control cultures containing equivalent amounts of histidine were incubated with the tester bacteria prior to plating out. Significant mutagenic activity was found in a high proportion of samples (18 of 20). The histidine content in gastric juice which can affect in vitro mutagenicity testing must be adequately controlled before positive or negative results can be equated with the presence or absence of intragastric carcinogens.

Adult↗
PubMed1984

An enzyme-linked immunosorbent procedure for assaying aflatoxin B1.

A polyclonal rabbit antibody preparation against aflatoxin B1 (AFB1), produced by immunization with a bovine serum albumin-AFB1 conjugate, has been used to develop an enzyme-linked immunosorbent assay (ELISA). AFB1-ovalbumin, obtained by reacting either AFB1-8,9-dichloride or -8,9-dibromide with ovalbumin, was used to coat each well of a polyvinyl microtitre dish. Rabbit antibody, diluted 1:100 000, was added to each well and left to attach for 90 min, then the excess was washed off with phosphate-buffered saline/Tween. Anti-rabbit IgG coupled to peroxidase was then added, left for 90 min and the excess washed away. Residual peroxidase activity was assayed using tetramethylbenzidine as substrate; the reaction was quenched at the end of the 15-min incubation period with 2 N sulfuric acid. Inhibitor studies to assay AFB1 and related compounds in urine involved prior incubation of the diluted anti-AFB1 antibody with inhibitor for 60 min at 37 degrees C, prior to dispensing into the multi-well plates. Inhibitor studies with AFB1 showed inhibition over a concentration range of 10(-1) to 10(-5) micrograms/ml. The minimum detectable concentration was approximately 10(-5) micrograms/ml (0.032 pmol/ml). Anti-AFB1 antibody was also inhibited by iro-AFB1-DNA, one of the forms of AFB1-DNA, as well as by AFB1-guanine. Undiluted urine from normal subjects inhibited antibody binding to plates; this inhibition could be prevented by either dilution or extraction procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗
PubMed1983

Improved conditions for the production of monoclonal antibodies to carcinogen-modified DNA, for use in enzyme-linked immunosorbent assays (ELISA).

The methodology for the production of monoclonal antibodies to chemical carcinogen-modified DNA has been improved to provide high yields of hybridomas, using guanine-imidazole ring-opened aflatoxin B1-modified DNA as an example (iro-AFB1 DNA). The percentage of immunised mice which responded to iro-AFB1 DNA-protein immunisation and the number of specific hybridomas produced was dependent on the level of modification of DNA. One in three BALB/c mice had detectable (but low) antibody titre when 0.3% modified iro-AFB1 DNA was used and this yielded 2 specific hybridomas, whereas all mice responded at reasonable titres and 6 specific hybridomas were obtained when 3% modified iro-AFB1 DNA was used. Other factors found to improve the number and titre of mice responding to immunisation and the yield of hybridomas were: KLH greater than BSA as carrier protein, C57 BL/6 X BALB/c F1 greater than BALB/c mice for antibody production, fusion success and ascites growth. The conditions limiting the sensitivity and reproducibility of an enzyme-linked immunosorbent assay (ELISA) using these monoclonal antibodies with beta-galactosidase-linked sheep F(ab')2 anti-mouse IgG as the second antibody were also tested. Present experience with AFB1 and other carcinogens indicates that these methods should be applicable to the production of monoclonal antibodies to DNA modified by a wide variety of chemical carcinogens.

Aflatoxin B1↗
PubMed1983

The biological activity of 4-chloromethylbiphenyl, benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity. A report of an individual study in the UKEMS genotoxicity trial 1981.

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial 'fluctuation' assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.

Animals↗
PubMed1983

Metabolic activation and bacterial mutagenicity of aflatoxin B1 in two different test systems.

The metabolism of aflatoxin B1 (AFB1) by liver post-mitochondrial supernatant fraction from phenobarbitone treated male albino rats in liquid suspension and soft-agar plate incorporation bacterial mutation assay systems has been studied. The AFB1 concentration used was in the range of those added to bacterial mutagenicity tests with this mycotoxin. Three oxidative metabolites of AFB1 viz the Tris derivative of AFB1 8,9-diol (derived from the 8,9-epoxide), aflatoxin Q1 (AFQ1) and M1 (AFM1) were observed. The metabolite profile and time course of formation were qualitatively similar in both assay systems. The rate and overall formation of metabolites in the soft-agar system was approximately one half that in the liquid suspension system which was reflected in decreased AFB1 induced bacterial mutation in the former system. The AFB1 metabolite profile in these in vitro systems did not mirror completely the reported in vivo profile seen in the rat, as aflatoxin P1 (AFP1) and a glutathione conjugate were not detected. No evidence was found for a prolongation of metabolism in the soft-agar system.

Aflatoxin B1↗
PubMed1983

Nitration of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons results in products able to induce transformation of Syrian hamster cells.

Five nitrated polycyclic aromatic hydrocarbons, synthesized from benzo[a]pyrene, fluoranthene, pyrene, and chrysene induced dose-dependent transformation of Syrian hamster embryo cells. Benzo[a]pyrene, a known carcinogen, induced transformation while the other parental compounds, which are non-carcinogens, were not effective. The transforming potential of the nitro derivatives varied from compound to compound; on a molar basis, 1,8-dinitropyrene was the most effective nitrated hydrocarbon followed in order by 3-nitrofluoranthene, 1-nitropyrene, 6-nitrochrysene, and 6-nitrobenzo[a]pyrene. The ability to obtain dose-dependent transformation frequencies indicates that hamster cell transformation represents a responsive model for elucidating the mechanism of action of nitrated carcinogens. Because of their ubiquitous distribution and their ability to induce morphological transformation in mammalian cells, nitrated polycyclic aromatic hydrocarbons must be considered as potential carcinogens.

Animals↗
PubMed1982

Macromolecular binding of [3H]6-nitrobenzo[a]pyrene in male rats.

Generally labelled [3H]6-nitrobenzo[a]pyrene (6NBP) when administered to male rats by i.p. injection was widely distributed throughout the body. After a single injection of 4.2 mg/kg the highest concentration was found in the liver (5% at 2 h) followed by the spleen greater than kidney greater than lung. Maximal covalent binding to macromolecules occurred between 8 and 24 h depending upon the organ; the order of binding/mg macromolecule was RNA greater than DNA greater than protein for all organs studied. In the liver maximal binding was found 8 h after 6NBP administration at which time there were 45 +/- 7 pmol 6NBP/mg bound to DNA and 114 +/- 40 pmol/mg to RNA following a dose of 4.2 mg/kg. These levels fall to 14 +/- 4 and 12 +/- 6 pmol/mg, respectively, after 168 h, the longest time point so far studied. At a lower dose of 1.4 mg/kg the values for DNA at 8 h were 14 +/- 2 pmol/mg and at 168 h 3 +/- 2 pmol/mg while for RNA the corresponding values were 29 +/- 8 and 15 +/- 3 pmol/mg. After gastric intubation at a dose of 4.2 mg/kg there was no difference in liver macromolecular binding at 24 h between conventional and germ-free animals. Enzymic digestion of liver DNA from [3H]6NBP injected animals and reversed-phase h.p.l.c. of the digests showed two peaks of radioactivity. The percentage of total radioactivity in each peak varied from preparation to preparation suggesting that one peak may be a degradation product of the other although experimental variability cannot be ruled out. The implications of these findings in relation to the potential carcinogenicity of 6NBP are discussed.

Animals↗
PubMed1982

Production of monoclonal antibodies to guanine imidazole ring-opened aflatoxin B1DNA, the persistent DNA adduct in vivo.

Monoclonal antibodiess were produced following immunisation of mice with guanine imidazole ring-opened aflatoxin B1 DNA (iro AFB1 DNA), coupled electrostatically to methylated keyhole limpet haemocyanin. Three monoclonal hybridoma lines producing antibodies specific for iro AFB1 DNA were grown as ascites tumours and suitable dilutions of the ascitic fluid (1:8000-1:50,000) used in a competitive enzyme linked immunosorbent assay (ELISA) to measure reactivity of the antibodies to a variety of aflatoxin and nucleic acid-related compounds. These antibodies recognise AFB1 bound to DNA at levels 10(4)-10(5) times lower concentration than unmodified calf thymus DNA or 8,9-dihydro-8,9-dihydroxy-aflatoxin B1: and show 2-5 times the affinity to iro AFB1 DNA compared to AFB1 DNA. The concentration of AFB1 in iro AFB1 DNA producing 50% inhibition in a competitive ELISA was 1.8 x 10(-7) molar. Using the most sensitive hybridoma line, levels of 1 adduct in 300,000 nucleotides would be detectable, which is the level of binding found in the rat and the hamster in vivo. These monoclonal antibodie should therefore prove useful in detecting these lesions in animal and human tissue samples exposed to aflatoxins.

Aflatoxin B1↗
PubMed1981

Nitrated polycyclic aromatic hydrocarbons: potent bacterial mutagens and stimulators of DNA repair synthesis in cultured human cells.

Ten polycyclic aromatic hydrocarbons (PAHs), viz. anthracene pyrene, chrysene, perylene, fluoranthene, benzo[a]pyrene, benzo[a]pyrene, benz[a]anthracene, benzo[ghi]perylene, benzo[k]fluoranthene, have been nitrated using concentrated nitric acid and the crude nitrated mixture examined for biological activity. All the nitro PAHs examined were mutagenic to Salmonella typhimurium in the absence of a rat liver preparation. Addition of Aroclor-1254 induced liver had little effect on mutagenicity. Mutagenic potency differed for the various nitrated mixtures with nitrated pyrene and nitrated fluoranthene the most potent and nitrated anthracene the least potent. Both frame-shift and base-substitution mutations were induced by the nitrated PAHs. The nitrated PAHs were also able to induce DNA repair synthesis in cultured HeLa cells in the absence of liver, indicating that these cells have the necessary enzymes to activate nitro PAHs. Potency again varied from compound to compound with nitrated pyrene appearing to be the most active. Isolation of individual components from the crude nitrated mixtures has not been carried out in this study. In view of the possible wide-spread distribution of nitrated PAHs in the environment further work is required to assess the carcinogenic potency of these compounds which possibly pose a risk to man.

Animals↗
PubMed1980

Some factors affecting mutant numbers in the Salmonella/microsome assay.

An examination has been made of some of the parameters which can affect mutant numbers in the Salmonella/microsome assay. The type of minimal media plates used for the assay and the concentration of glucose-6-phosphate, one of the co-factors necessary for mono-oxygenase action, had no effect on mutant numbers. Increases in mutated bacteria resulted from the use of (1) log-phase bacteria, (2) higher NADP concentrations than those normally recommended, and (3) higher phosphate buffer concentrations. Six mutagens, i.e., 2-acetylaminofluorene (AAF), 3,3'-dichlorobenzidine (3,3'-DCB), cyclophosphamide (CY), aflatoxin B1 (AFB1), 3-methylcholanthrene (3MC) and benzo[a]pyrene (BP), all requiring mono-oxygenase activation, were studied with two Salmonella typhimurium strains, TA98 and TA100, and liver preparations from rats given different inducing agent treatments using optimum conditions. Phenobarbitone induction was generally superior to Aroclor-1254 in converting these substrates to mutagens except for the polycyclic hydrocarbon substrates. A comparison of 3-methylcholanthrene, Aroclor-1254, beta-naphthoflavone or phenobarbitone as inducing agents revealed the first three of these to be equally effective in activating BP or 3MC to mutagens, whereas phenobarbitone was less active. Dual administration of 3-methylcholanthrene and phenobarbitone to rats did not result in an additive mutagenic effect using AAF, AFB1 or 3,3'-DCB as substrates, the numbers of mutant bacteria obtained being only equal to that seen with 3-methylcholanthrene alone. These differences were not due to there being different liver protein optima for the various inducing agent treatments. The foregoing results are discussed in relation to attempts to draw up a rigid protocol for mutagenicity testing.

Animals↗
PubMed1980

A high pressure liquid chromatography study on the removal of DNA-bound aflatoxin B1 in rat liver and in vitro.

Following intraperitoneal administration of [3H] aflatoxin B1 (AFB1) to young adult male rats, there is rapid uptake of the carcinogen by the liver, the target organ for carcinogenesis, leading to DNA covalent binding. Acid hydrolysis of this DNA shows that after 2h, the major DNA adduct is trans 8,9-dihydro-8-(7-guanyl)-9-hydroxy AFB1 (AFB1-gua). By 24h after AFB1 administration the major DNA adduct is no longer AFB1-gua but a product with the identical retention time on h.p.l.c. to 8,9-dihydro-8-(N5-formyl-2',5',6' triamino-4' oxo-N5-pyrimidyl)-9-hydroxy AFB1 (AFB1-triamino-Py). 48h after carcinogen administration, only a small amount of AFB1-gua remains and the major product is AFB1-triamino-Py. The half-life of removal of AFB1-gua is 22h, while AFB1-triamino-Py is much more persistent. In vitro incubation studies on DNA isolated from rats treated 2h previously with [3H] AFB1 show that at pH 7.4 AFB1-gua is the major product released from the DNA with some release of 8,9-dihydro-8,9-dihydroxy AFB1, (AFB1-diol). If more extensively reacted AFB1-DNA is used than that obtained from in vivo administration, then the rate of AFB1-diol release is enhanced while that of AFB1-gua is reduced. It would appear, therefore, that much of the release of AFB1 from DNA in vivo within the first 24h is probably not through a DNA repair process but through chemical release arising from the positively charged N7-guanine. There is considerable conversion of AFB1-gua to AFB1-triamino-Py on in vitro incubation of DNA as well as AFB1-gua and AFB1-diol release. By 24h approximately 66% of the bound AFB1 is in the form of AFB1-triamino-Py and after 48h the conversion is complete. The complex pattern of AFB1-release from DNA may have important consequences in both the induction of mutations and in tumour initiation.

Aflatoxin B1↗
PubMed1979

Carcinogen prediction in the laboratory: a personal view.

Although carcinogens can be divided into various categories, i.e. viruses, physical agents and synthetic and naturally occurring chemicals, it is the latter that give rise to the greatest concern because of their number, quantity and distribution. Present methods of testing chemicals for potential carcinogenicity rely in the main on administration of these at maximally tolerated dose levels to animals, usually rodents, for the animals' lifetime. Such tests would be economically impractical for all chemicals to which man is exposed. New methods have recently been introduced to screen large numbers of chemicals quickly and cheaply which rely on the unifying hypothesis that all carcinogenic chemicals are electrophiles or must be converted to such by metabolism. These methods will be reviewed and compared with traditional methods of carcinogenicity testing, particularly as to their role in attempting to predict hazard to man.

Alkylating Agents↗