Development of structure-activity relationship rules for predicting carcinogenic potential of chemicals.

Since the inception of Section 5 (Premanufacturing/Premarketing Notification, PMN) of the Toxic Substances Control Act (TSCA), structure-activity relationship (SAR) analysis has been effectively used by U.S. Environmental Protection Agency's (EPA) Structure Activity Team (SAT) in the assessment of potential carcinogenic hazard of new chemicals for which test data are not available. To capture, systematize and codify the Agency's predictive expertise in order to make it more widely available to assessors outside the TSCA program, a cooperative project was initiated to develop a knowledge rule-based expert system to mimic the thinking and reasoning of the SAT. In this communication, we describe the overall structure of this expert system, discuss the scientific bases and principles of SAR analysis of chemical carcinogens used in the development of SAR knowledge rules, and delineate the major factors/rules useful for assessing the carcinogenic potential of fibers, polymers, metals/metalloids and several major classes of organic chemicals. An integrative approach using available short-term predictive tests and non-cancer toxicological data to supplement SAR analysis has also been described.

Assessment of carcinogenic hazard of chemical mixtures through analysis of binary chemical interaction data.

Assessment of the potential health hazard of environmental complex chemical mixtures is one of the most difficult and challenging problems in toxicology. In this article, we describe the development of an innovative computerized system for ranking and predicting potential cancer hazard of chemical mixtures. We take into consideration both the additive risk of individual carcinogens present and the projected overall interaction effect of the mixture based on analyzing and integrating the possible interaction effects of all binary pairs of individual constituents of the mixture. Using this system, it can be predicted that a number of mixtures of polycyclic aromatic hydrocarbons should have a carcinogenic risk lower than that calculated by the simple additivity model, whereas the reverse is true for a number of other mixtures. The system can be very useful in hazard ranking and priority setting in dealing with mixture problems such as cleanup of hazardous waste.

Comparative pulmonary tumorigenesis in DBA/2J and C57Bl/6J mice by administration of 3-methylcholanthrene and dimethylnitrosamine singly and combined.

C57Bl/6J mice, which are inducible for both hepatic and pulmonary aryl hydrocarbon hydroxylase (AHH), and DBA/2J mice, which are noninducible for hepatic AHH but moderately inducible for pulmonary AHH, received dimethylnitrosamine (DMN) i. p., or methylcholanthrene (MCA) orally, or a combination of both agents, for 10 weeks; the animals were observed for an additional 26 weeks. The dosing schedule and total dose of DMN or MCA or DMN + MCA received were identical to those used by other investigators in their syncarcinogenesis bioassay study in Swiss mice. The lung lesions induced in the present experiments were alveologenic tumors and adenomatosis. Alveologenic tumors were induced in a much larger number of DBA/2J mice than in C57Bl/6J mice (87.9% versus 14.0%). If, however, the sum of alveologenic tumors and adenomatous nodules is considered and is expressed per lung lesion bearing mouse, then these ratios are for the control, MCA, DMN and MCA + DMN groups: 2.7, 1.5, 3.5, and 5.4 in the DBA/2J mice and 1.0, 1.3, 2.7, and 3.6 in the C57Bl/6J mice, respectively. This suggests a relatively greater susceptibility of the C57Bl/6J strain if the ratios are compared to the respective control values. This greater susceptibility of the C57Bl/6J is best seen by comparing the percent increase of the ratio for the MCA + DMN groups; the net increase is 100% for DBA/2J and 260% for C57Bl/6J. In the DBA/2J strain more extrapulmonary tumors (hemangioendotheliomas, liver and kidney tumors) were induced than in the C57Bl/6J strain by DMN or MCA + DMN, but not by MCA alone.

Comparative effects of indole and aminoacetonitrile derivatives on dimethylnitrosamine-demethylase and aryl hydrocarbon hydroxylase activities.

The effect of in vivo administration of indole and five 3-indolyl derivatives including L-tryptophan, as well as of aminoacetonitrile and 3 of its derivatives, were studied on the carcinogen-metabolizing hepatic mixed-function oxidases dimethylnitrosamine (DMN)-demethylase I and II and aryl hydrocarbon hydroxylase (AHH). Indole, 3-indolylmethanol, 3-indolyl-acetonitrile, 3-indolylacetone and L-tryptophan induce AHH activity from 3- to 6-fold of the control level, whereas beta-3-indolylethanol has no effect; the latter compound produces a 21% decrease of the endoplasmic reticulum content in the tissue. Only L-tryptophan induces DMN-demethylase I and only L-tryptophan and 3-indolylmethanol induce DMN-demethylase II, representing a doubling of enzyme activity in all 3 instances. Aminoacetonitrile is a potent repressor of DMN-demethylase I. Substitutions on the amino group bring about strong decrease or abolishment of mixed-function oxidase repressor activity; thus, iminodiacetonitrile has only about 1/5th the repressor activity of the parent compound, whereas nitrilotriacetonitrile and dimethylaminoacetonitrile appear to be inactive. Aminoacetonitrile and its derivatives studied have no effect on DMN-demethylase II and AHH activities. The mixed-function oxidase-modifying effects of the indole compounds and of aminoacetonitrile and its derivatives illustrate the potential complexity of effects of dietary constituents on the carcinogenic responses.

Interaction of the tobacco-specific nitrosamines, methylethylnitrosamine and N-nitrosonornicotine, with DNA and guanosine.

In vitro binding of the tobacco-specific nitrosamines, methylethylnitrosamine (MEN) and nitrosonornicotine (NNN), to exogenous DNA and guanosine was studies in a rat liver microsome-catalyzed system. MEN (N-[ethyl-1-14C]) binds covalently to calf thymus DNA whereas NNN (N'-[pyrrolidine-2-14C]) binds only to guanosine but not to DNA. Pretreatment of the rats with either phenobarbital (PB) or 3-methylcholanthrene (MC) greatly diminishes the binding of 14C-MEN to DNA. Both MEN-demethylase and -deethylase activities are stimulated by PB pretreatment and inhibited by MC pretreatment, but the degree of stimulation and inhibition of the two dealkylases are not the same. Addition of cytosol to the incubation system does not enhance but suppresses the binding of 14C-MEN to DNA. Inclusion of mitochondria in the system has no effect on the binding. Addition of benzylamine (250 microM), which is a potent inhibitor of dimethylnitrosamine-demethylase, however totally abolishes the binding of 14C-MEN catalyzed by microsomes. The data suggest that ethylcarbonium ion may be the metabolically activated intermediate of MEN that binds to DNA.

Enhancement of toxicity and enzyme-repressing activity of p-dioxane by chlorination: stereoselective effects.

The acute toxicity of p-dioxane may be enhanced up to 1000-fold by chlorination of the compound. The effect was stereoselective. Of the stereoisomers tested, tetrachloro-p-dioxane, isomer I (2r, 3t, 5t, 6c) was over 80 times more toxic than isomer II (2r, 3c, 5t, 6t). The latter compound was also a potent repressor of hepatic dimethylnitrosamine-demethylase I (DMN-d) and aryl hydrocarbon hydroxylase (AHH).

Role of dimethylnitrosamine-demethylase in the metabolic activation of dimethylinitrosamine.

In vivo administration to rats of the mixed-function oxidase modifiers 3-methylcholanthrene (MC), pregnenolone-16 alpha-carbonitrile (PCN) or beta-naphthoflavnoe (beta-f) inhibits the hepatic microsome-catalyzed in vitro binding of dimethylnitrosamine (DMN) to DNA. This parallels their effect on DMN-demethylase I, regarded to be the sole activating step in DMN carcinogenesis and fails to account for the previously observed anomaly that MC and PCN inhibit, while beta-NF enhances, the hepatocarcinogenic activity of DMN. The in vitro binding of DMN is clearly dependent on microsomes and NADPH, and is strongly enhanced by soluble cytoplasmic proteins; the presence of the latter has no effect. however, on the relative response to pretreatment by the modifiers. In mice beta-NF enhances and PCN inhibits DMN-demethylase I; beta-NF has no effect on either the cytochrome P-450 level or on the LD50, while PCN strongly increases the cytochrome P-450 level but without influencing the LD50. Neither of the two modifiers has any effect in mice on the host-mediated mutagenicity of DMN in a dose-response study, except for the highest dose of DMN (200 mg/kg) where PCN pretreatment significantly enhanced mutagenicity. To account for the anomalous observations, other potential pathways of DMN metabolism have been explored. Whole rat liver nuclei or isolated nuclear membrane fractions contain no DMN-demethylase or diethylnitrosamine-deethylase activity. In a microsomal mixed-function amine-oxidase assay system neither purified enzyme preparations nor whole microsomes catalyze NADPH oxidation in the presence of DMN as substrate. In addition, the purified enzyme does not catalyze formaldehyde production in the DMN-demethylase assay system. Benzylamine, a typical inhibitor of mitochondrial monoamine oxidase (MAO), is a potent inhibitor of DMN-demethylase activity, but microsomes are devoid of MAO activity. Furthermore, purified MAO has no DMN-demethylase activity. The differential effect of modifiers on the carcinogenicity of DMN probably involves pathways other than DMN metabolism.

Differential effects of beta-naphthoflavone and pregnenolone-16alpha-carbonitrile on dimethylnitrosamine-induced hepatocarcinogenesis.

The effect of administration of beta-naphthoflavone (beta-NF) or pregnenolone-16alpha-carbonitrile (PCN) on the hepatocarcinogenicity of dimethylnitrosamine (DMN) in male SD rats was explored. Both beta-NF and PCN are potent repressors of the low Michaelis constant enzymatic form of DMN-demethylase, a mixed-function oxidase that catalyzes DMN demethylation. DMN-induced hepatocarcinogenesis was inhibited by PCN and was enhanced by beta-NF. Seven liver tumors were found in 45 rats fed DMN plus PCN compared to 14 liver tumors in 43 rats fed DMN alone; 32 liver tumors were found in 43 rats fed DMN plus beta-NF. No liver tumors were detected in rats that received only PCN, beta-NF, or the administration vehicles. Of the 53 liver tumors observed, 53% were angiosarcomas; this type of tumor was found in all 3 groups of rats that received DMN.

Ultrastructural and metabolic determinants of resistance to azo-dye susceptibility to nitrosamine carcinogenesis of the guinea-pig.

During diethylnitrosamine (DEN) administration, a distinctive difference was observed between rats and guinea-pigs in the sequence of ultrastructural changes in the hepatic endoplasmic reticulum (ER). In DEN-induced hepatic tumour cells in the guinea-pig there was extensive proliferation of the rough ER, while the smooth ER was quite sparse; in the premalignant liver the opposite was noted. This is in contrast to the rat, in which administration of either DEN or 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) brings about, in both premalignant and malignant hepatic tissue, proliferation of the smooth ER and sparsity of the rough ER. Yet, as in the rat, the number of ribosomes on the outer surface of the guinea-pig liver rough ER is greatly reduced and this is paralleled by a 49% decrease of the RNA/protein ratio as early as 4 weeks of nitrosamine administration. The decrease of RNA/protein ratio and ultrastructurally observed loss of ribosomes from the ER, following nitrosamine administration, correlate with a decrease of photometric response of microsomal suspensions to the sulphydryl probe, p-chloromercuribenzoate. While azo-dye-reductase activity is higher in untreated rats than in untreated guinea-pigs, feeding 3'-Me-DAB for 6 weeks brings about a 76% decrease in the rat, but no significant decrease in the guinea-pig, which is refractory to azo-dye carcinogenesis. Thus, the ability of the liver to inactivate the dye is greatly decreased in the rat, but not in the guinea-pig, as administration progresses toward the threshold dose for tumorigenesis. On the other hand, constitutive levels of nitrosamine dealkylase are identical in the 2 species and remain essentially unchanged following administration of DEN for 10 weeks. Inasmuch as nitrosamine dealkylation represents activating metabolism, this provides a rationale for the comparable susceptibility of the rat and guinea-pig to DEN carcinogenesis. Of the 2 enzymes in the 2 species, it is only azo-dye reductase in the guinea-pig which appears to be unregulated by glucose repression, since starvation brings about no change in this activity. Starvation-induced increase of azo-dye reductase in the rat is not influenced by administration of 3'-Me-DAB and only slightly by DEN. The starvation-induced increase of nitrosamine dealkylation is abolished, however, in both species by administration of DEN but only slightly decreased by 3'-Me-DAB.

Structural identification of p-dioxane-2-one as the major urinary metabolite of p-dioxane.

Analysis by gas chromatography (GC) of the volatile compounds present in the urine from rats administered dioxane, a hepatic carcinogen to this species, revealed a major metabolite. The appearance of the metabolite was pH-dependent, undetectable at high pH; reacidification of the urine sample brought about the reappearance of the metabolite. The amount excreted was dose-dependent and time-dependent, reaching a maximum between 20 and 28 h after dioxane administration. Diethylene glycol administered to rats gave rise to the same metabolite. When isolated and purified from lyophilized urine by preparative GC, the metabolite exhibited an intense carbonyl band at 1750 cm-1 in the infrared spectrum. Nuclear magnetic resonance spectrum showed two triplets and one singlet with equal intensity at delta 3.85, 4.48 and 4.37, respectively. GC-mass spectrometric studies indicated a parent peak at m/e 102. The metabolite was identified as p-dioxane-2-one. Synthetic reference compound exhibited identical IR, NMR, and GC-mass spectra as the metabolite. The tentative pathway and the biological significance of dioxane metabolism are discussed.