Acrylonitrile, an industrial chemical, is a multisite carcinogen in rats and mice, producing tumors in four tissues with barrier function, that is, brain, forestomach, Zymbal’s gland, and Harderian gland. To assess mechanism(s) of action (MoA) for induction of neoplasia and to evaluate whether the findings in rodents are indicative of human hazard, data on the potential key effects produced by acrylonitrile in the four rodent target tissues of carcinogenicity were evaluated. A notable finding was depletion of glutathione in various organs, including two target tissues, the brain, and forestomach, suggesting that this effect could be a critical initiating event. An additional combination of oxidative DNA damage and cytotoxic effects of acrylonitrile and its metabolites, cyanide, and 2-cyanoethylene oxide, could initiate pro-inflammatory signaling and sustained cell and tissue injury, leading to compensatory cell proliferation and neoplastic development. The in vivo DNA-binding and genotoxicity of acrylonitrile has been studied in several target tissues with no compelling positive results. Thus, while some mutagenic effects were reported in acrylonitrile-exposed rodents, data to determine whether this mutagenicity stems from direct DNA reactivity of acrylonitrile are insufficient. Accordingly, the induction of tumors in rodents is consistent primarily with a non-genotoxic MoA, although a contribution from weak mutagenicity cannot be ruled out. Mechanistic data to support conclusions regarding human hazard from acrylonitrile exposure is weak. Comparison of metabolism of acrylonitrile between rodents and humans provide little support for human hazard. Three of the tissues affected in bioassays (forestomach, Zymbal’s gland, and Harderian gland) are present only in rodents, while the brain is anatomically different between rodents and humans, diminishing relevance of tumor induction in these tissues to human hazard. Extensive epidemiological data has not revealed causation of human cancer by acrylonitrile.

Chemicals with carcinogenic activity in either animals or humans produce increases in neoplasia through diverse mechanisms. One mechanism is reaction with nuclear DNA. Other mechanisms consist of epigenetic effects involving either modifications of regulatory macromolecules or perturbation of cellular regulatory processes. The basis for distinguishing between carcinogens that have either DNA reactivity or an epigenetic activity as their primary mechanism of action is detailed in this review. In addition, important applications of information on these mechanisms of action to carcinogenicity testing and human risk assessment are discussed.

Genotoxicity of flavor and fragrance materials was assessed in Turkey Egg Genotoxicity Assay (TEGA) using 32P-nucleotide postlabeling (NPL) and comet assays to detect hepatic DNA adducts and strand breaks. Twenty materials having results in GADD45a-Gluc 'BlueScreen HC' genotoxicity assay, and standard in vitro and in vivo tests, were selected to evaluate the accuracy of TEGA. Quinoline (QUI) and 2-acetylaminofluorene (AAF) served as positive comparators. Two materials, p-tert-butyldihydrocinnamaldehyde (BDHCA) and methyl eugenol (MEU) produced DNA adducts. BDHCA, p-t-butyl-α-methylhydrocinnamic aldehyde (BMHCA), trans-2-hexenal (HEX) and maltol (MAL) produced DNA strand breaks. Fifteen other materials were negative in both assays. Based on reports of oxidative DNA damage induction by MAL and 4-hydroxy-2.5-dimethyl-3(2H) furanone (HDMF), modified comet assays were conducted. Positive comet findings for MAL were not confirmed, and only equivocal evidence of oxidative damage was found. Accordingly, MAL was judged to have equivocal genotoxicity in TEGA. HDMF was positive in modified comet assay, indicating an ability to produce oxidative DNA damage. TEGA showed modest concordance with results in regulatory in vitro assays. Findings in TEGA, with few exceptions, were concordant with the results of in vivo genotoxicity and carcinogenicity testing. Thus, TEGA is an attractive alternative model for the assessment of genotoxic potential of chemicals in vivo.

The Chicken Egg Genotoxicity Assay (CEGA) demonstrated responsiveness to various DNA-reactive chemicals requiring metabolic activation, which implies broad bioactivation capability. To assess potential metabolic competence, expression profiles of metabolic genes in the embryo-chicken fetal liver were determined using microarray technology. Fertilized chicken eggs were injected under the CEGA protocol with vehicle (deionized water [DWD, the activation-dependent carcinogens, diethylnitrosamine (DEN), and N-nitrosodiethanolamine (NDELA) at doses producing no effect on survival. Previously in CEGA, DEN produced DNA damage, whereas NDELA did not. Expressions of 463 genes known to encode for phase I and II of endo- and xenobiotic metabolism were detected on the array. DW did not affect the expression of the selected genes, deregulating less than 1% of them. In contrast, DEN at 2 mg/egg and NDELA at 4 mg/egg produced significant transcriptomic alterations, up-regulating up to 41% and down-regulating over 31% of studied genes. Both nitrosamines modulated the majority of the genes in a similar manner, sharing 64 up-regulated and 93 down-regulated genes with respect to control group, indicating similarity in the regulation of their metabolism by avian liver. Differences in gene expression between DEN and NDELA were documented for several phase I CYP 450 genes that are responsible for nitrosamine biotransformation, as well as for phase II genes that regulate detoxication reactions. These findings could underlie the difference in genotoxicity of DEN and NDELA in CEGA. In conclusion, the analysis of gene expression profiles in embryo chicken fetal liver dosed with dialkylnitrosamines demonstrated that avian species possess a complex array of inducible genes coding for biotransformation.

Exposure to acrylonitrile induces formation of tumors at multiple sites in rats, with females being more sensitive. The present study assessed possible mechanisms of acrylonitrile tumorigenicity, covalent DNA binding, DNA breakage, and oxidative DNA damage, in two target tissues, the brain and Zymbal's glands, of sensitive female Fischer (F344) and Sprague-Dawley (SD) rats. One group received acrylonitrile in drinking water at 100 ppm for 28 days. Two other groups were administered either acrylonitrile in drinking water at 100 ppm or drinking water alone for 27 days, followed by a single oral gavage dose of 11 mg/kg bw 14C-acrylonitrile on day 28. A positive control group received a single dose of 5 mg/kg bw of 7-14C-benzo[a]pyrene, on day 27 following the administration of drinking water for 26 days. Using liquid scintillation counting, no association of radiolabeled acrylonitrile with brain DNA was found. In accelerator mass spectrometry analysis, the association of 14C of acrylonitrile with DNA in brains was detected and was similar in both strains, which may reflect acrylonitrile binding to protein as well as to DNA. Nucleotide 32P-postlabeling assay analysis of brain samples from rats of both strains yielded no evidence of acrylonitrile DNA adducts. Negative conventional comet assay results indicate the absence of direct DNA strand breaks in the brain and Zymbal's gland in both strains of rats dosed with acrylonitrile. In both rat strains, positive results in an enhanced comet assay were found only in brain samples digested with formamidopyrimidine-DNA glycosylase but not with human 8-hydroxyguanine-DNA glycosylase, indicating possible oxidative DNA damage, other than 8-oxodG formation. In conclusion, definitive evidence of DNA binding of acrylonitrile in the brain and Zymbal's gland was not obtained under the test conditions. A role for oxidative stress in tumorigenesis in the brain but not Zymbal's gland may exist.

Male rats are more susceptible to the induction of liver cancer by the aromatic amine 2-acetylaminofluorene (AAF) than are females. To assess the basis for this difference and to determine whether sex differences in susceptibility to AAF are present in human liver cells, the DNA reactivity of AAF was measured in livers of male and female Sprague–Dawley (SD) rats and in cultured SD rat and human hepatocytes of both sexes. In livers of rats administered oral doses of AAF, the total levels of adducts measured by nucleotide postlabelling at up to 8 weeks were about twofold greater in males than in females. Similarly, the level of AAF-DNA adducts formed in cultured male rat hepatocytes dosed with AAF was about twofold greater than in female rat hepatocytes. Also, the level of DNA repair synthesis was about threefold greater in AAF-dosed cultured male rat hepatocytes compared with female, indicating that the greater adduct levels in males was not due to lesser repair. In contrast, in cultured human hepatocytes of both sexes, AAF produced similar levels of adducts and DNA repair synthesis, which were intermediate between those produced in male and female rat hepatocytes. Thus, the greater susceptibility of male rats to AAF hepatocarcinogenicity is due at least in part to greater bioactivation and formation of AAF-DNA adducts in hepatocytes. Moreover, the data from human hepatocytes suggest that human liver could be less susceptible than male rat liver to the carcinogenic effects of aromatic amine carcinogens of the AAF type.

The use of a food substance is Generally Recognized as Safe (GRAS) through scientific procedures or experience based on common use in food. The pivotal data used for GRAS determination must be of common knowledge and should include evidence for safety under the conditions of intended use of the substance. Such evidence includes data on the identity and specifications of the substance, its properties of absorption, distribution, metabolism and excretion, and depending on the level of concern, data on genotoxicity, acute and subchronic toxicity, reproductive and developmental toxicity and carcinogenicity. Several alternative procedures can be used as the replacement for standard scientific procedures in order to improve the GRAS process.

Administration of AAF by gavage to male rats resulted in formation of several DNA adducts in liver, the pattern of which changed over time and with repair. The cumulative dose of 0.125 mg kg−1 AAF achieved in repeated doses for 16 weeks was a NOAEL for DNA adducts.

Purpose : The emergence of prostate cancer as a major health issue and the absence of curative treatment for metastatic disease requires the development of new treatment modalities. Prostate specific antigen (PSA) and prostate-specific membrane antigen (PSMA) are possible targets for prostate cancer immunotherapy. We have previously shown that PSA and PSMA can be expressed in recombinant bacille Calmette-Guerin (BCG) strains. Methods : The in vivo immunogenicity of the prostate-specific proteins produced by this recombinant BCG strain were examined by detection of specific antibody responses and delayed-type hypersensitivity (DTH) responses in mice vaccinated with these strains. These immune responses were compared with those of control mice vaccinated with phosphate buffered saline diluent or soluble PSA or PSMA. Results : Mice vaccinated with rBCG-PSA developed low levels of anti-PSA antibodies and strong DTH to PSA. Mice vaccinated with rBCG-PSMA developed strong DTH to PSMA and no anti-PSMA antibodies. Conclusions : We conclude that recombinant BCG expressing PSA or PSMA induce strong cellular immune responses to these antigens. We propose that the innate adjuvant capacity of BCG could help stimulate a specific immune response against prostate-specific proteins produced by the bacteria , which in turn , could lead to the eradication of undetected metastatic prostate cancer cells in post-surgical patients.

A panel of pathologists (Panel) was formed to evaluate the pathogenesis and human relevance of tumors that developed in the fundic region of rat stomachs in carcinogenicity and mechanistic studies with alachlor and butachlor. The Panel evaluated stomach sections stained with hematoxylin and eosin, neuron-specific enolase, and chromogranin A to determine the presence and relative proportion of enterochromaffin-like (ECL) cells in the tumors and concluded all tumors were derived from ECL cells. Biochemical and pathological data demonstrated the tumor formation involved a nongenotoxic threshold mode of action (MOA) initially characterized by profound atrophy of the glandular fundic mucosa that affected gastric glands, but not surface epithelium. This resulted in a substantial loss of parietal cells and a compensatory mucosal cell proliferation. The loss of parietal cells caused a marked increase in gastric pH (hypochlorhydria), leading to sustained and profound hypergastrinemia. The mucosal atrophy, together with the increased gastrin, stimulated cell growth in one or more ECL cell populations, resulting in neoplasia. ECL cell autocrine and paracrine effects led to dedifferentiation of ECL cell tumors. The Panel concluded the tumors develop via a threshold-dependent nongenotoxic MOA, under conditions not relevant to humans.

The alkenylbenzene methyleugenol occurs naturally in a variety of spices and herbs, including basil, and their essential oils. At high dose levels methyleugenol induces hepatocarcinogenicity in rodents following bioactivation to 1'-sulfooxymethyleugenol which forms DNA adducts. This study investigated whether the inhibitory effect of the basil flavonoid nevadensin on sulfotransferase (SULT)-mediated bioactivation of methyleugenol observed in vitro would also be reflected in a reduction of DNA adduct formation and a reduction in an early marker for liver carcinogenesis in an 8-week rat study. Co-exposure to methyleugenol and nevadensin orally resulted in a significant inhibition of liver methyleugenol DNA adduct formation and in inhibition of hepatocellular altered foci induction, representing indicators for initiation of neoplasia. These results suggest that tumor formation could be lower in rodent bioassays when methyleugenol would be dosed in a matrix containing SULT inhibitors such as nevadensin compared to experiments using the pure methyleugenol.

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens.

The potential of the carcinogenic tobacco specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-1-butanone (NNK) to induce preneoplastic hepatocellular altered foci (HAF) was tested in the in ovo carcinogenicity assessment (IOCA) assay. Single doses of NNK over a dose range from 0.1 mg to 6 mg were injected into fertilized turkey eggs prior to incubation for 24 days. The livers were investigated by histological, histochemical and morphometric methods. Mortality was increased for eggs exposed to 6 mg. In this group, the whole livers were severely altered, showing pronounced changes of nucleus size and signs of cell death. At the dose of 2 mg various types of foci of altered hepatocytes (HAF) were observed. Basophilic cell foci of the solid or tubular type were most frequent. The NNK-induced HAF were very similar to the preneoplastic lesions that occur in the livers of mammals during hepatocarcinogenesis which are regarded as early indicators of carcinogenesis. The similarity to the HAF in rodents included histochemically detectable alterations like decreased activities of glucose-6-phosphatase, adenosine triphosphatase and glycogen phosphorylase. At doses of 1 mg or below, no HAF were detected. At all dose levels an increased occurrence of enlarged hepatocytes with enlarged nuclei and prominent nucleoli (karyomegalic hepatocytes) were observed. The increase in karyomegalic hepatocytes was also statistically significant at the low dose of 0.1 mg/kg NNK but the dose–effect curve for their induction was clearly non-linear. Induction of HAF and karyomegalic hepatocytes in ovo is a simple (one dose), rapid (24 days) and inexpensive (no animal purchase or housing) experimental approach for studies on chemically induced hepatocarcinogenesis.

Methyleugenol (MEG), a constituent of plants used in the human diet, is hepatocarcinogenic in rodents. In an experiment to elucidate its mode of action in rat liver, male F344 rats were administered MEG intragastrically at 3 doses per week for up to 16 weeks in an initiation phase, after which half the rats were fed 500 ppm phenobarbital (PB) in the diet to promote liver neoplasia and the other half were maintained on control diet for 24 weeks. At 8 and 16 week interim terminations, 32P-nucleotide postlabeling assay revealed 3 adducts in livers of all MEG groups. The hepatocellular replicating fractions, measured by proliferating cell nuclear antigen immunohistochemistry, were doubled or more in all MEG groups. Hepatocellular altered foci, detected by glutathione S-transferase-placental type (π) immunohistochemistry, were present beginning with the high dose group at 8 weeks and extending to all MEG groups at 16 weeks. At the end of maintenance/promotion phase, the incidences, multiplicity and size of foci was similar between control and low dose groups, while those of mid and high dose groups were increased. Hepatocellular adenomas occurred in the mid and high dose groups, attaining higher multiplicity and size with PB. Thus, MEG had rapid initiating activity, reflecting the formation of DNA adducts and possibly cell proliferation.

Central to the concepts of thresholds or virtually safe doses for chemical carcinogensis their mode of action (MoA). One MoA entails epigenetic or non-DNA-reactive mechanisms, which do not involve direct reaction of the chemical with target cell DNA but rather produce cellular effects such as indirect genotoxicity or enhanced cell proliferation. Epigenetic carcinogens are generally accepted to have cancer thresholds at exposures below which they do not elicit the cellular effects in target organs that lead to carcinogenicity. In contrast, DNA-reactive or genotoxic carcinogens operate by a MoA that includes direct reactions with DNA in target tissues to form chemical-specific adducts resulting in mutations in critical genes. Mutagenesis and carcinogenesis can therefore theoretically be initiated by a single hit at a critical target in the DNA of a single target cell, but biological processes exist which ameliorate the genotoxicity of DNA-reactive carcinogens. Most studies of DNA-reactive carcinogens that used relative low cumulative doses (CDs) have been interpreted as not showing a threshold, although some investigators have calculated ‘virtually safe doses’, ‘practical thresholds’ or ‘no-adverse-effect-levels’(NOAEL). In this review of dose–effect relationships for DNA-reactive liver carcinogens we define thresholds as CDs below which there are no-effect-levels (NOELs), and as being distinct from NOAELs, values below which there are no observable adverse (carcinogenic) effects. We discuss dose–effect data for DNA-reactive hepatocarcinogens, including DNA adducts, cell proliferation and preneoplastic lesions, with regard to possible threshold effects. We conclude that NOAELs for liver tumour initiation exist.

The in ovo carcinogenicity assessing (IOCA) assay was used to examine the morphological changes in fetal turkey livers caused by the DNA-reactive carcinogen diethylnitrosamine (DEN). Fertilized turkey eggs were allocated into 3 groups: nondosed control (NDC), vehicle (water) control (VC) and DEN-dosed. At day 0, the fertilized eggs of the dosed groups were injected with 1 (LD) or 4 (HD) mg/egg (about 12.5 or 50 mg/kg egg) of DEN and the VC were injected with water. All eggs were allowed to incubate at 37°C and 60% humidity for 24 days. The fetal livers were collected and processed for histopathological evaluation (H&E staining). Typical survival rates were 82% for the NDC, 50% for the VC and 16–65% for the DEN-dosed fetuses. No difference in histology was found between NDC and VC control groups. Both DEN concentrations produced dose-related liver findings consisting of foci of altered hepatocytes (FAH), which had assumed a tubular cord (glandular) pattern, and in HD DEN group the FAH assumed a tumor-like morphology. In addition, the high DEN dose produced gallbladder agenesis. Thus, DEN produced both hepatocellular transformation (FAH) similar to preneoplastic microscopic changes in adult rodents, reflecting disruption of the fetal processes of differentiation and proliferation, and also teratogenicity (gallbladder agenesis).

Ethyl acrylate (EA) is an acrylic monomer used in the manufacture of a variety of polymers and copolymers as components of many commercially important products. Human exposure to EA occurs primarily in the workplace via inhalation or dermal contact. In F344 rat and B6C3F1 mouse studies of EA carcinogenicity conducted by the National Toxicology Program [National Toxicology Program, NTP, 1986. Carcinogenesis Studies of Ethyl Acrylate (CAS No. 140-88-5) in F344/N Rats and B6C3F1 Mice (Gavage Studies) (Tech. Rep. Ser. No. 259; NIH Publication No. 87-2515), Research Triangle Park, NC, USA], the only increased tumor incidences was in squamous cell papillomas and carcinomas of the forestomach, when EA was administered by gavage in corn oil at 100 or 200 mg/kg/day (high dose; HD). The neoplasms were preceded by forestomach irritation, inflammation, hyperkeratosis and hyperplasia of the forestomach mucosa. In studies in which rats and mice were exposed at comparable doses to EA in drinking water, by inhalation, or by dermal application, no neoplasms in the forestomach or in any other tissue were reported. EA exhibited clastogenicity and related mutagenicity in vitro, but was non-genotoxic in vivo, including in the forestomach of treated rats. The in vitro clastogenicity response correlates well with cellular toxicity, mediated by non-protein sulfhydryl depletion and mitochondrial impairment. Thus, the carcinogenicity in the forestomach can be ascribed to a non-genotoxic mode of action (MOA). The forestomach mucosal hyperplastic and even dysplastic changes, observed chronically, were reversible, provided the HD exposure was not longer than 6 months. This again supports a non-genotoxic MOA. In addition, the route and rate of EA exposure in rodents for forestomach neoplasia are irrelevant to potential human exposure, since humans do not have forestomach and are not exposed to EA by oral bolus. Thus, the weight of evidence indicates that the tumors produced in the rodent carcinogenicity studies arise from conditions that are irrelevant for human risk assessment.