Carl N. Martin

Aflatoxin B-oxide generated by chemical or enzymic oxidation of aflatoxin B1 causes guanine substitution in nucleic acids

AFLATOXIN B1 (AFB1) has the highest biological activity of the four naturally occurring aflatoxins produced by the mould Aspergillus flavus1. Feeding studies have shown it to be the most potent liver carcinogen known for the rat2. Its ingestion by man is associated with the high primary liver cancer incidence in certain parts of Africa3. There is much evidence to suggest that AFB1 requires metabolism to exert both its carcinogenic and mutagenic effects (reviewed in ref. 4). Structure-activity5, as well as chemical studies6,7 indicate that the major route for activation proceeds through mixed function oxidase attack yielding the 8,9-oxide (previously called the 2,3-oxide but renumbered according to IUPAC recommendations). Attempts to chemically synthesise this metabolite have so far been unsuccessful. We report here that peracid oxidation of AFB1 generates AFB1-8,9-oxide, and that this latter compound reacts readily with nucleic acids to give adducts identical to those obtained after liver mixed function oxidase (MFO) activation in vitro and in vivo.

Mutagenicity of methyl-, ethyl-, propyl- and butylnitrosourea towards Escherichia coli WP2 strains with varying DNA repair capabilities.

Methyl- (MNUA), ethyl- (ENUA), propyl- (PNUA) and butylnitrosourea (BNUA) have been tested for toxicity and mutation in a liquid suspension assay towards Escherichia coli WP2 and some of its repair deficient derivatives. A comparison of survival rates after nitrosourea exposure between WP2 and WP2 uvrA showed no difference between the two strains but a consistent difference in potency between the various nitrosoureas studied. Toxicity increased in the order MNUA less than PNUA less than ENUA less than BNUA. ENUA and PNUA induced a greater number of trp+ revertants in both strains than did MNUA and BNUA, particularly at low survival rates. None of these differences in biological potency could be accounted for by differences in rates of hydrolysis. ENUA, PNUA and BNUA were non-mutagenic towards WP2 lexA, WP2 recA and WP2 uvrA lexA, whereas MNUA did induce mutations. Ethyl methanesulphonate (EMS) was able to mutate WP2 lexA. These results are discussed in the light of current theories regarding the mechanism of action of these compounds.

N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine, the putative major DNA adduct of cyclophosphamide in vitro and in vivo in the rat

The anti-cancer agent, cyclophosphamide, metabolises to the cytotoxic alkylating agent phosphoramide mustard, which can be dephosphoramidated to give nornitrogen mustard. A rat liver mitochondrial supernatant system was used to study the binding of [chloroethyl 3H]cyclophosphamide to DNA. The reacted DNA was acid-hydrolysed and one major adduct was identified using Sephadex G-10 chromatography, followed by HPLC, using reversed-phase or ion-exchange systems. Further studies, using [14C]guanine as reaction substrate for [chloroethyl 3H]cyclophosphamide, phosphoramide mustard or nornitrogen mustard, demonstrated the main adduct from each reaction had identical chromatographic properties in these systems. The radiolabelled ratio in the [3H]cyclophosphamide-[14C]guanine reaction demonstrated a monoadducted product. From this evidence and from 1H NMR data, the common adduct was putatively identified as a hydroxylated nornitrogen mustard adduct (N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine). In in vivo studies, rats were injected intraperitoneally with 2.775 MBq [3H]cyclophosphamide. Total organ [3H] content and DNA binding levels were ascertained. Maximal levels of [3H] binding to DNA were seen between 1-4 hr with the highest binding levels observed in the bladder. The in vivo adduct was shown, using various HPLC systems, to co-chromatograph with the in vitro adduct and thus the main in vivo adduct was putatively identified as N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine.

Comparative lung tumorigenicity of parent and mononitro-polynuclear aromatic hydrocarbons in the BLU:Ha newborn mouse assay

A BLU:Ha newborn mouse lung adenoma bioassay was employed to compare the tumorigenicity of selected mononitroarenes and unsubstituted parent compounds 6 months after initial treatment. The presence of a nitro group had a variable effect upon compound potency in which tumorigenicity was increased, abolished, or unchanged. On the basis of results with equimolar doses, the potency of benzo[a]pyrene was greater than 6-nitrobenzo[a]pyrene (inactive), 6-nitrochrysene was much greater than chrysene (inactive), 3-nitrofluoranthene (active) was equal to fluoranthene (active), and 1-nitropyrene (inactive) was equivalent to pyrene (inactive). The potency series among the mononitroarenes was 6-nitrochyrsene much greater than 3-nitrofluoranthene greater than 6-nitrobenzo[a]pyrene (inactive) = 1-nitropyrene (inactive). Lung tumor incidence and multiplicity were similar for both males and females. No consistent pattern was observed for the occasional appearance of lymphoma or hepatic nodular hyperplasia in the various treatment groups.

Measurement of ‘unscheduled’ DNA synthesis in HeLa cells by liquid scintillation counting after carcinogen treatment

HeLa cells, conditioned in an arginine-deficient medium to reduce DNA S-phase synthesis, were treated with one of four ultimate carcinogens (MNNG, BrMBA, N-acetoxy AAF and EMS) and one precarcinogen, AFB1. All treated cells preferentially incorporated [3H] thymidine as a result of DNA repair monitored by liquid scintillation counting of the extracted DNA. The cells showed some capacity to activate AFB1, but repair synthesis was much increased if a rat liver mixed function oxidase preparation was also present. At equimolar concentrations the various carcinogens stimulated different amounts of DNA repair; this variation was not proportional to the carcinogenic potency of the chemicals tested. Reasons for this are discussed as is the use of this technique as a screen for chemical carcinogens.

The use of isolated rat hepatocytes to measure unscheduled DNA synthesis as a screen for chemical carcinogens

Isolated rat hepatocytes are being used in a variety of ways to answer fundamental questions concerning the metabolism of chemical compounds. These cells retain a high capacity to metabolise xenobiotics when treated shortly after isolation, as either suspensions or after attachment. The advantage of intact cells over rat liver enzyme preparations such as post-mitochondrial supernatant in assessing the likely in vivo metabolic fate of xenobiotics are numerous. When unscheduled DNA synthesis (UDS) induced in these cells is used as an endpoint to detect electrophile generation by carcinogens, again important questions concerning the extent and route of activation can be answered. However, the detection of UDS in these cells has been suggested as a possible screen for the detection of chemical carcinogens. It is the very advantage of the system in approaching the true in vivo situation that may work against its usefulness as such a screen by reducing its sensitivity.

Comparison of the Mutagenic Potency of DNA Adducts Formed by Reactive Derivatives of Aflatoxin, Benzidine and 1-Nitropyrene in a Plasmid System

1-Nitropyrene is an ubiquitous environmental pollutant which has been detected in the urban atmosphere in many areas of the world (1). It is generated primarily by incomplete combustion of fossil fuels and thus is present in relatively high concentrations in emissions such as diesel engine exhaust fumes (2, 3) and power station fly-ash (4, 5). This compound has been shown to be biologically active in a number of in vitro genotoxicity assay systems, inducing mutation in Salmonella typhimurium (6) and inducing mutations, unscheduled DNA synthesis, transformation or chromosomal rearrangements in a variety of mammalian cell lines (7–10). 1-nitropyrene has also been shown to be carcinogenic in laboratory animals (11, 12). The activation of 1-nitropyrene has been shown to involve a sequential two-electron reduction leading to the formation of 1-NOP, N-OH-l-aminopyrene and 1-aminopyrene (13, 14). The N-hydroxy derivative has been shown to be an ultimate DNA-reactive species (13). The adduct produced in DNA by the reactive metabolite of 1-nitropyrene has been identified in bacterial systems (15) and in rat liver (16) as N-(deoxyguanosin-8-yl)-1-aminopyrene. Thus, 1-nitropyrene is a compound to which the human population is exposed and is biologically active in a number of experimental systems. However, very little information is available as to the relative carcinogenic risk to humans chronically exposed to this compound.

A Comparison of the Tissue Distribution and Covalent Binding to Hepatic DNA of [3H]Benzidine and [3H]4,4″-Diaminoterphenyl in the Rat

4,4″-Diaminoterphenyl (DAT) appeared to be unlike benzidine (BZD) in that N-acetylation may not be necessary for the production of a reactive species capable of covalent interaction with DNA. The data generated showed that acetyl CoA supplementation, whilst significantly increasing the mutagencity of BZD in S. typhimurium TA98, has at best only a marginal effect on the activity of DAT. BZD was readily N-acetylated by addition of acetic anhydride in vitro, whereas DAT was refractory to acetylation by this agent. BZD binds to the C-8 atom of guanosine in DNA in vivo to yield N-(deoxyguanosin-8-yl)-N′-acetylbenzidine which is susceptible to de-acetylation by carboxylesterase enzymes. The DNA adduct induced in rat liver DNA in vivo by DAT however remained unchanged following treatment by carboxylesterase. These data, in combination with other results reported here, suggest the structure of the DAT-DNA adduct may be N-(deoxyguanosin-8-yl)DAT although no physio-chemical analysis was performed. Tissue distribution of DAT was qualitatively similar to that found for BZD, however, was quantitatively only 50% of the BZD level measured as percentage of dose administered. Binding of DAT to rat liver DNA 24 h after i.p. injection was approximately one-fifth the level observed following injection of the same dose of BZD.