Limitations of metabolic activation systems used with in vitro tests for carcinogens.

Important differences between the metabolic activation of 7,12-dimethylbenz[a]anthracene in intact cellular systems and in liver homogenates suggest that the use of homogenates in conjunction with short-term assays for carcinogens could yield misleading results.

Sequence specificity in the interaction of the four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides with the supF gene.

The shuttle vector pS189 was treated with each of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide, and the modified DNA was used as a template in a polymerase arrest assay examining the supF gene. Sites at which polymerase (Sequenase, version 2.0) progress along the template was blocked were presumed to be at or near sites of adduct formation. The polymerase arrest sites were compared with recently reported mutation hotspots induced by these agents in this gene (Bigger et al., Proc. Natl. Acad. Sci. USA, 89: 368-372, 1992). For 31 of 32 mutation hotspots, a polymerase arrest band was present at or 1 or 2 nucleotides 3'- to that site, indicating that adduct formation tended to be associated with mutation hotspots. However, the arrest bands near mutation hotspots were not particularly prominent in all cases, and there were many sites of substantial polymerase arrest that were not in the vicinity of mutation hotspots. Thus, factors in addition to chemical selectivity must play key roles in determining sites of mutation.

Evaluation of metabolic activation of 7,12-dimethylbenz(a)anthracene in vitro by aroclor 1254-induced rat liver S-9 fraction.

Short-term assays for detection of chemical carcinogens frequently rely on an Aroclor 1254-induced rat liver S-9 fraction for metabolic activation of test compounds. The ability of this in vitro system to reproduce the activation occurring in target tissue was investigated by examining the DNA adducts produced when the polycyclic aromatic hydrocarbon carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), was incubated with the S-9 fraction and calf thymus DNA. Analyses by Sephadex LH-20 column chromatography of hydrocarbon-deoxyribonucleoside adducts obtained after enzymic digestion of the [3H]DMBA-modified DNA revealed that the products of binding of DMBA to DNA in the presence of the S-9 fraction vary with the relative concentration of DMBA to S-9 fraction. Further analyses of these adducts by high-pressure liquid chromatography in the presence of the diol-epoxide-DNA adduct (isolated from mouse embryo cells exposed to [14C]DMBA) and chemically synthesized ultraviolet-absorbing markers of DMBA 5,6-oxide-deoxyribonucleoside adducts showed that, at high DMBA-S-9 ratios, DMBA 5,6-oxide-deoxyriboiucleoside adducts were prominent among the products while, at low DMBA-S-9 ratios, the products included the diol-epoxide-DNA adduct found in target tissue. However, this adduct was always accompanied by other adducts not found in intact cellular systems. Inclusion of a metabolic inhibitor (1,1,1-trichloropropylene oxide) in the Salmonella mutagenicity assay demonstrated that high levels of revertants can be obtained from rat liver S-9 fraction-activated DMBA under conditions which should prohibit formation of the diol-epoxide. These results suggest that Aroclor 1254-induced rat liver S-9 fraction does not exactly reproduce the metabolic activation of this particular carcinogen in vivo and therefore should not be assumed to do this for other carcinogens.

Products of binding of 7,12-dimethylbenz(a)anthracene to DNA in mouse skin.

7,12-Dimethylbenz(a)anthracene (DMBA):deoxyribonucleoside-adducts, from enzymatic hydrolysis of DNA from mouse skin exposed to [3H]DMBA in vivo, were analyzed by reverse-phase high-pressure liquid chromatography. Double-labeling studies showed that the adducts were qualitatively identical to those formed in mouse embryo cell cultures. These have been tentatively identified as bay-region anti-dihydrodiol epoxide: deoxyguanosine- and :deoxyadenosine adducts and a bay-region syn-dihydrodiol epoxide:deoxyadenosine-adduct (where the terms syn and anti define dihydrodiol-epoxides wherein the benzylic hydroxyl group and epoxide oxygen are cis or trans to one another, respectively). The relative amounts of individual adducts did not vary substantially with time or with the sex of the mice. However, the syn-dihydrodiol-epoxide:deoxyadenosine-adduct did increase with dose and constituted as much as 40% of the total DNA binding at high doses of DMBA. This is in contrast to the much lower (2 to 3%) levels of binding to deoxyadenosine residues in mouse skin reported for the less potent tumor initiator benzo(a)pyrene. The greater reactivity of DMBA with deoxyadenosine residues in mouse skin may play a role in determining its greater tumor initiating potential.

Comparison of metabolism-mediated binding to DNA of 7-hydroxymethyl-12-methylbenz(a)anthracene and 7, 12-dimethylbenz(a)anthracene.

Comparison of the binding to DNA of 7-hydroxymethyl-12-methylbenza(a)anthracene and 7, 12-dimethylbenz(a)anthracene (DMBA) catalyzed by mouse embryo cells in culture or by rat liver microsomes indicates that the products formed are different for the two hydrocarbons. Thus, the hydroxy compound is not an intermediate in the binding of DMBA to DNA in these systems. Binding of the hydroxy compound to DNA in mouse embryo cells is less efficient than for DMBA and is inhibited by 1,1,1-trichloropropylene 2,3-oxide, an inhibitor of epoxide hydrase. This and the fluorescence spectra of the hydroxy compound-DNA adducts indicate that the hydroxy compound is activated for DNA binding through the formation of a diol-epoxide in the 1,2,3,4-ring. As previously found for DMBA, this is consistent with the activation of this compound through a bay-region diol-epoxide.

Genetic toxicology assessment of HI-6 dichloride.

The oxime HI-6 dichloride [1-(2 hydroxyiminomethyl -1-pyridino)-3-(4-carbamoyl-1-pyridino)-2-oxapropane dichloride monohydrate] has shown to be a potent reactivator of cholinesterase activity and may have efficacy for the treatment of organophosphate intoxication [SIPRI, 1976; Schenk et al.; Arch Toxicol 36:71-81, 1976]. As part of a preclinical safety assessment program, the genetic toxicology of HI-6 dichloride was evaluated in a series of assays designed to measure induction of gene mutations and chromosomal aberrations. HI-6 dichloride gave negative responses in the Salmonella mutagenicity assay and in the CHO/HGPRT gene mutation assay. Dose-dependent increases in the frequency of chromosomal aberrations were noted when HI-6 dichloride was tested in cultured CHO cells and in cultured human peripheral blood lymphocytes. The mouse lymphoma gene mutation assay, reputed to measure both gene mutations and chromosomal deletions, was negative in the absence of metabolic activation. Depending on the criteria employed, a negative or equivocal response was seen in the presence of rat liver-derived S-9 mix. An in vivo rat bone marrow metaphase assay performed to further investigate the in vitro clastogenic responses was negative. The results from these studies indicate that HI-6 dichloride does not induce gene mutations in vitro; however, it is clastogenic in vitro but does not appear to be clastogenic in vivo.

Mechanism of action of food-associated polycyclic aromatic hydrocarbon carcinogens.

The polycyclic aromatic hydrocarbon carcinogens are formed in the inefficient combustion of organic matter and contaminate foods through direct deposition from the atmosphere or during cooking or smoking of foods. These potent carcinogens and mutagens require metabolism to dihydrodiol epoxide metabolites in order to express their biological activities. In vitro studies show that these reactive metabolites can react with the bases in DNA with different specificities depending upon the hydrocarbon from which they are derived. Thus, the more potent carcinogens react more extensively with adenine residues in DNA than do the less potent carcinogens, with the result that mutation at A . T base pairs is enhanced for the more potent carcinogens. In the past few years, considerable clarification of the mechanism of metabolic activation have been achieved and the focus for the immediate future is expected to be on how the reactive metabolites actually bring about biological responses.

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene.

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5, 6-dimethylchrysene, of benzo[g]chrysene and of 7-methylbenz[a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at A&z.ccirf;T or G&z.ccirf;C pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[a]pyrene or benzo[c]phenanthrene dihydrodiol epoxide-DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.

Influence of adrenalectomy and sex on the binding of 3-methylcholanthrene to cytosol macromolecules of rat liver.

The retention of 3-methylcholanthrene (3-MC) in rat hepatic cytosol was significantly enhanced by adrenalectomy. In contrast, there was no significant difference in 3-MC retention in females as compared with males. 3-MC present in the cytosol fraction was bound to macromolecules and could be separated into three fractions by ion-exchange column chromatography.

Variation in route of microsomal activation of 7,12-dimethylbenz [a]anthracene with substrate concentration +.

The nature of the metabolites of 7,12-dimethylbenz[a]anthracene (DMBA) binding to DNA in the presence of Aroclor-induced rat liver microsomes is not affected by the presence or absence of magnesium ions, but is dependent on the concentration of the hydrocarbon. At high concentrations of DMBA, the primary route of metabolic activation is through the K-region oxide while, at low concentrations of DMBA, activation is through other routes. A small proportion of these latter products elute from Sephadex LH-20 columns with mouse embryo cell [14C]DMBA-DNA adducts which are known to arise through reaction of the bay region diol-epoxide of DMBA with cellular DNA. In contrast to the dose-dependence of activation of DMBA by microsomes, the binding of this carcinogen to DNA in intact cultured mouse embryo cells is not qualitatively influenced by concentration over a forty-fold dose range. These findings suggest limitations in the use of microsomal systems as models for target tissue activation.

DNA polymerase action on bulky deoxyguanosine and deoxyadenosine adducts.

In order to determine how individual hydrocarbon-DNA adducts give rise to specific mutations, a single-stranded oligonucleotide, 5'-T8GT10AT8C2T4CT3CT-3', was reacted with the carcinogen 7-bromomethylbenz[a]anthracene which generates both deoxyguanosine and deoxyadenosine adducts in DNA. The products were separated by HPLC to yield unmodified oligonucleotide and oligonucleotide modified either at the single guanine, or at the single adenine, residue. Incubation of these products with 32P-5'-end-labeled primer, 5'-AGA3GA4G2-3', modified T7 DNA polymerase (Sequenase) and deoxyribonucleoside-5'-triphosphates followed by gel electrophoretic analysis indicated that unmodified oligonucleotide template allowed the primer to be rapidly extended to give species of the same length as the template (40 nucleotides) and of 41 nucleotides in length. However, primer extension for the templates containing the guanine and adenine adducts was held up initially (1 min) at the nucleotide preceding the adduct. At longer times (up to 15 min) a nucleotide was added opposite the adduct and, to a lesser extent, another nucleotide was added beyond this. Some full-length oligonucleotide was also synthesized with these carcinogen-modified templates. When synthesis was allowed to proceed only to the nucleotide preceding the adduct, and this template-extended primer complex incubated with individual nucleotide triphosphates plus Sequenase, it was found that deoxyadenosine residues were most readily incorporated opposite the adduct irrespective of whether it was a deoxyguanosine or deoxyadenosine adduct. These results, which suggest that G.C----T.A and A.T----T.A transversions would be the mutagenic consequences of formation of bulky hydrocarbon adducts at guanines and adenines respectively, are consistent with the most frequent hydrocarbon-induced mutational changes reported thus far.

Preferential mutagenesis at G.C base pairs by the anti 3,4-dihydrodiol 1,2-epoxide of 7-methylbenz[a]anthracene.

The racemic anti-dihydrodiol epoxide of 7-methylbenz[a]anthracene preferentially induced mutations at G.C base pairs in the pS189 shuttle vector. Mutations were not randomly distributed throughout the supF target gene, but were concentrated at five hotspots. The hotspots for this agent did not correspond exactly to those produced by any other dihydrodiol epoxide examined to date, indicating that dihydrodiol epoxide structure and reactivity play a major role in determining mutagenic hotspots.

Mutagenic specificities and adduct distributions for 7-bromomethylbenz[a]anthracenes.

Mutation induction in the supF gene of the plasmid pS189 by 7-bromomethylbenz[a]anthracene and 7-bromomethyl-12-methylbenz[a]anthracene was examined. The former compound was substantially more mutagenic than the latter but a much greater proportion of the total mutations were located at mutation hotspots for the 12-methyl derivative. The overall correlation between sites of mutation and sites of polymerase arrest (an indicator of adduct formation) through the supF gene was poor. Although these bromocompounds should form only a single guanine adduct (unlike dihydrodiol epoxides that form both cis and trans adducts) more than one mutational change was found at a given site, although the predominant base substitution was G-->T for either compound.

Mutagenic specificity of a potent carcinogen, benzo[c]phenanthrene (4R,3S)-dihydrodiol (2S,1R)-epoxide, which reacts with adenine and guanine in DNA.

Mutations were induced in the supF gene of the pS189 shuttle vector by treatment with optically active benzo[c]phenanthrene (4R,3S)-dihydrodiol (2S,1R)-epoxide in vitro and replication in human cells. The induced mutation frequency was 60-fold greater than the spontaneous rate, and most of the mutations analyzed were transversions (86%), which principally consisted of similar numbers of A.T----T.A and G.C----T.A changes. The unusual susceptibility of A.T pairs to mutation by this chemical agent is consistent with its chemical reactivity toward adenine and argues that the mutations are targeted to the adducts formed. The central base in the sequences 5'-AGA-3', 5'-AAC-3', and 5'-GAG-3' was particularly susceptible to mutation. Twelve "hotspots" in the supF gene accounted for most mutations seen. Some of these hotspots differed from those found by others for racemic benzo[a]pyrene dihydrodiol epoxide and, even when a hotspot was common, the mutagenic changes were not always the same. Although adenine insertion opposite a noninstructional lesion could account for most of the data, no single mutagenic mechanism could encompass all of it. The cellular machinery that converts chemical damage to mutations must determine the mutational result to a large extent, but the findings herein show that the chemical agent itself plays a large role in determining both the location and the nature of the mutations that arise.

Mutagenic specificities of four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides.

The pS189 shuttle vector carrying a supF target gene was used to compare the mutagenic specificities of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide. One of these isomers is the most tumorigenic dihydrodiol epoxide tested to date and another is essentially inactive as a tumorigen. Overall mutagenicities were not correlated with tumorigenicities, but each configurational isomer induced a unique spectrum of mutational hot spots in the supF target gene, which monitors primarily point mutations. It is suggested that the demonstrated isomer-specific selectivity for mutation targets within the supF gene may be indicative of a similar selectivity for one gene versus another and that such selectivity may be one determinant of relative tumorigenicity.

7,12-dimethylbenz[a] anthracene--DNA binding in mouse skin: response of different mouse strains and effects of various modifiers of carcinogenesis.

7,12-Dimethylbenz[a]anthracene (DMBA)--deoxyribonucleoside adducts formed in mouse skin DNA were quantified in order to determine whether these changed in any systematic fashion under conditions where the tumorigenic activity of DMBA is modified. Similar distributions of adducts were found in male NIH Swiss mice and C57BL mice which exhibit different sensitivities to initiation-promotion using DMBA as initiator, though in both these strains of mice the bay region syn dihydrodiol epoxide is responsible for a greater fraction of total binding at higher DMBA doses. Pretreatment with various chemicals known to inhibit the tumor initiating activity of DMBA in mouse skin did not lead to selective inhibition of the formation of any adduct in female NIH Swiss mice. However, the effects of these agents ranged from a clear inhibition of overall DNA binding (7,8-benzoflavone) to little or no effect on overall binding (butylated hydroxyanisole, butylated hydroxytoluene). The lack of any effect of the antioxidants on DMBA--DNA adduct formation suggests that they may affect some step in tumor initiation other than adduct formation.

Mutational specificity of the anti 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene.

An SV40-based pS189 shuttle vector, which contained a supF target gene and was replicated in human cells (Ad293), was used to determine the mutational specificity of anti 5-methylchrysene 1,2-dihydrodiol 3,4-epoxide, the active metabolite of the environmentally prevalent carcinogen 5-methylchrysene. The frequency of supF mutants containing point mutations increased with dose to approximately 40 times the spontaneous frequency. The induced mutations were not randomly distributed but occurred preferentially at mutagenic hotspots, which were not all identical to those reported by others for benzo[a]pyrene dihydrodiol epoxide, a metabolite with similar chemistry.