In vivo short-term assays for tumor initiation and promotion in the glandular stomach of Fischer rats

Here we summarize the data on 55 compounds tested in in vivo short-term assays for tumor-initiating and tumor-promoting activity in the glandular stomach of male Fischer (F344) rats. Most of the data has been previously published. Tumor-initiating activity was assayed by measuring the induction of unscheduled DNA synthesis (UDS) and DNA single strand scission; tumor-promoting activity was assayed by measuring the induction of ornithine decarboxylase (ODC) activity, increased replicative DNA synthesis (RDS), and of c-fos and c-myc oncogene expression. The compounds were orally administered. Twenty-nine compounds were tested for UDS. Eight were positive, including 5 glandular stomach carcinogens; 16 were negative, including 5 liver carcinogens; and 5 were equivocal. Twenty compounds were tested for DNA single strand scission. Twelve were positive, including 6 glandular stomach carcinogens; 7 negative, including 2 liver carcinogens; and 1 was equivocal. Thirty-two compounds were tested for RDS. Twenty-six were positive, including 8 glandular stomach carcinogens and 6 glandular stomach tumor-promoters; 4 were negative, including 3 liver carcinogens and a stomach irritant; and 2 were equivocal. Forty-five compounds were tested for ODC. Thirty-seven were positive, including 8 glandular stomach carcinogens and 6 glandular stomach tumor promoters; 7 were negative, including 3 liver carcinogens; and one was equivocal. All glandular stomach carcinogens and tumor-promoters examined were positive in both RDS and ODC. Two compounds were tested for c-fos and c-myc expression; one was a glandular stomach carcinogen and one was a glandular stomach tumor promoter, and both were positive. In addition, 2 compounds inhibited the increase in RDS induced by the tumor promoter NaCl, suggesting anti-tumor-promoter activity. Thus these assays are useful for assessing potential tumor-initiating and tumor-promoting activity in the rat glandular stomach.     

Yeast DEL assay detects clastogens

Chromosomal rearrangements, including DNA deletions are involved in carcinogenesis. The deletion (DEL) assay scoring for DNA deletions in the yeast Saccharomyces cerevisiae is able to detect a wide range of carcinogens. Among approximately 60 compounds of known carcinogenic activity, the DEL assay detected 86% correctly whereas the Ames Salmonella assay detected only 30% correctly [R.J. Brennan, R.H. Schiestl, Detecting carcinogens with the yeast DEL assay, Methods Mol. Biol. 262 (2004) 111-124]. Since the DEL assay is highly inducible by DNA double strand breaks, this study examined the utility of the DEL assay for detecting clastogens. Ten model compounds, with varied mechanisms of genotoxicity, were examined for their effect on the frequency of DNA deletions with the DEL assay. The compounds tested were: actinomycin D, camptothecin, methotrexate and 5-fluorodeoxyuridine, which are anticancer agents, noscapine and furosemide are therapeutics, acridine, methyl acrylate and resorcinol are industrial chemicals and diazinon is an insecticide. The in vitro micronucleus assay (IVMN) in CHO cells, a commonly used tool for detection of clastogens, was performed on the same compounds and the results of the two assays were compared. The results of our study show that there is 70% concordance in the presence of metabolic activation (rat liver S9) and 80% concordance in the absence of metabolic activation between the DEL assay and the standard in vitro micronucleus assay. The lack of cytotoxicity observed for four of the ten compounds examined indicates limited diffusion of lipophilic compounds across the yeast cell wall. Thus, the development of a more permeable yeast tester strain is expected to greatly improve concordance of the DEL assay with the IVMN assay. The yeast DEL assay is inexpensive, amenable to automation and requires less expertise to perform than the IVMN. Thus, it has a strong potential as a robust, fast and economical screen for detecting clastogens in vitro.     

Mutagenicity of aliphatic nitrosamines in Salmonella typhimurium.

25 aliphatic nitrosamines were examined in the Ames assay for bacterial mutagens, using rat liver "S-9" for activation. Of them, 8 carcinogens were mutagenic and 5 non-carcinogens were not mutagenic. However, 2 compounds not carcinogenic in rats were mutagenic and 9 carcinogens were not mutagenic, including 6 that are liver carcinogens in rats.     

Evaluation of the DNA-repair host-mediated assay. I. Induction of repairable DNA damage in E. coli cells recovered from liver, spleen, lungs, kidneys, and the blood stream of mice treated with methylating carcinogens

The DNA-repair host-mediated assay was further calibrated by determining the genotoxic activities of 4 methylating carcinogens, namely, dimethylnitrosamine (DMNA), 1,2-dimethylhydrazine (SDMH), methyl nitrosourea (MNU) and methyl methanesulphonate (MMS) in various organs of treated mice. The ranking of the animal-mediated genotoxic activities of the compounds was compared with that obtained in DNA repair assays performed in vitro. The differential survival of strain E. coli K-12/343/113 and of its DNA-repair-deficient derivatives recA, polA and uvrB/recA, served as a measure of genotoxic potency. In the in vitro assays and at equimolar exposure concentrations, MMS and MNU are the most active chemicals, followed by DMNA, which shows a slight genotoxic effect only in the presence of mouse liver homogenate; SDMH has no activity under these conditions. In the host-mediated assays, the order of genotoxic potency of the compounds was quite different: those carcinogens which require mammalian metabolic activation, namely, DMNA and SDMH, show strong effects in liver and blood, a lesser effect in the lungs and kidneys and the least effect in the spleen. The activity of MNU, a directly acting compound, is similar in all organs investigated, but it is clearly lower than that of DMNA and SDMH. MMS, also a directly acting carcinogen, causes some (barely significant) effect at the highest dose tested. A similar order of potency was observed when the compounds were tested in intrasanguineous host-mediated assays with gene mutation as an endpoint. DMNA and SDMH induce comparable frequencies of L-valine-resistant mutants in E. coli K-12/343/113 recovered from liver and spleen of treated mice, the effect in the liver being the strongest. MNU is mutagenic only at a higher dose, while MMS shows no effect. The results are discussed with respect to the literature data on organ-specific DNA adduct formation induced by the compounds. It is concluded that qualitatively there is a good correlation between the degree of genotoxic activity found in the DNA repair host-mediated assay and DNA adduct formation in the animal's own cells. This is exemplified by the finding that the relative order of genotoxic activity of the 4 methylating agents in bacteria recovered from various organs (DMNA approximately equal to SDMH greater than MNU greater than MMS) is reflected by the same order of magnitude in DNA alkylation in corresponding mammalian organs. Quantitatively, the indirectly acting agents DMNA and SDMH seem to induce fewer genotoxic effects in bacteria present in the liver than would be expected on the basis of DNA-adduct formation data.     

Mutagenicity of aliphatic nitrosamines in Salmonella typhimurium

25 aliphatic nitrosamines were examined in the Ames assay for bacterial mutagens, using rat liver “S-9” for activation. Of them, 8 carcinogens were mutagenic and 5 non-carcinogens were not mutagenic. However, 2 compounds not carcinogenic in rats were mutagenic and 9 carcinogens were not mutagenic, including 6 that are liver carcinogens in rats.     

Aerobic and anaerobic reduction of nitrated pyrenes in vitro

Nitrated pyrenes are mutagenic and tumorigenic environmental pollutants that are activated to DNA-binding derivatives via nitroreduction. We have investigated the enzymatic nitroreduction of 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene to determine if differences in the extent of nitroreduction may help explain differences in their biological potencies. Each nitrated pyrene was incubated aerobically and anaerobically with 105,000 X g supernatant (S105) from Salmonella typhimurium TA98 and the nitroreductase-deficient strain, TA98NR, and with cytosol and microsomes from rat and human liver. Under anaerobic conditions, 1-nitropyrene and 1,3-dinitropyrene were reduced by TA98 S105 to a lesser extent than 1,6- and 1,8-dinitropyrene. The extent of 1,6- and 1,8-dinitropyrene metabolism was not altered relative to TA98 when using TA98NR S105, but the nitroreduction of 1-nitropyrene and 1,3-dinitropyrene was decreased. Both rat and human liver cytosol and microsomes reduced 1,6- and 1,8-dinitropyrene to greater extents than 1-nitropyrene and 1,3-dinitropyrene. Under aerobic conditions rat and human liver cytosols were similar to TA98 S105 in that aminopyrene decreased while nitrosopyrene formation increased. By comparison, oxygen decreased the microsomal formation of both nitrosopyrenes and aminopyrenes. The reduction of succinoylated cytochrome c was measured during the hepatic metabolism of nitro- and nitrosopyrenes under aerobic conditions. The data indicated that reduced nitro- and nitrosopyrene intermediates were directly reducing succinoylated cytochrome c and that the assay could be used as a measure of aerobic nitroreduction. These studies demonstrate that 1,6- and 1,8-dinitropyrene are reduced to a greater extent than 1-nitropyrene and 1,3-dinitropyrene, which corresponds to their relative biological potencies as mutagens and carcinogens. Furthermore, although more extensive nitroreduction is detected under anaerobic conditions, the nitroreduction that occurs aerobically may be important for the mutagenic and tumorigenic properties of these compounds.     

Evaluation of the DNA repair host-mediated assay. II. Presence of genotoxic factors in various organs of mice treated with chemotherapeutic agents

The DNA repair host-mediated assay was further calibrated by testing 7 chemotherapeutic agents known to possess carcinogenic activity, namely bleomycin (BLM), cis-diamminedichloroplatinum-II (cis-Pt), cyclophosphamide (CP), diethylstilboestrol (DES), isonicotinic acid hydrazide (isoniazid, INH), natulan (NAT) and mitomycin C (MMC). Differential survival of wild-type and uvrB/recA E. coli strains served as a measure of genotoxic activity. In in vitro assays, BLM, cis-Pt and MMC exhibited high genotoxic activity. The other 4 compounds had no measurable effect on the survival of the two strains, either with or without mouse liver preparations. In the host-mediated assays BLM, cis-Pt, MMC and also NAT induced strong killing of the DNA repair-deficient bacteria recovered from liver, spleen, lungs, kidneys and the blood of treated mice compared to the wild-type strain. The results are not indicative of large organ-specific differences in genotoxically active amounts of the drugs immediately after their application to the host animals. CP, INH and DES did not show geneotix activity in these assays even at very high exposure levels. To compare the genetic endpoint measured in the DNA repair assays, i.e. induction of repairable DNA damage, with the induction of gene mutations, the ability of the 7 drugs to induce valine-resistant (VALr) mutants in E. coli was measured in host-mediated assays under identical treatment conditions. INH showed considerable mutagenic activity in E. coli cells recovered from liver and spleen, while BLM and MMC induced a 3-4-fold increase in VALr mutants above spontaneous levels. The other compounds showed no mutagenic activity under these in vivo conditions. From these results it can be concluded that the type of primary DNA lesions produced by these chemotherapeutic agents (cross-links by MMC and cis-Pt, and strand breaks by BLM and possibly by NAT; base alkylation by INH) appears to determine whether a compound will be highly positive in the DNA repair assay as in the case of BLM, cis-Pt, MMC and NAT, and less effective in inducing mutations under similar conditions, or whether the opposite will occur, as in the case of INH; DES and CP probably do not interact sufficiently with bacterial DNA to show an effect in either of the genetic endpoints; and the present DNA repair host-mediated assay may represent a sensitive, rapid and economic method for monitoring genotoxic factors in various organs of experimental animals which have been treated with cytostatic drugs.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo

There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus (MN) test (traditionally the most widely-used test) is negative. Not all rodent carcinogens give positive results in the micronucleus test, and so it has been common practice to include a second in vivo assay such as the unscheduled DNA synthesis (UDS) test. This has proved useful but is usually limited to analysis of rodent (usually rat) liver. With the increased evaluation and use of other in vivo assays, e.g. for transgenic mutations (TG) and DNA damage (Comet assay) it was important to investigate their usefulness. We therefore examined the published in vivo UDS, TG and Comet-assay results for 67 carcinogens that were negative or equivocal in the micronucleus test. Between 30 and 41 chemicals were evaluated in each of the three in vivo tests, with some overlap. In general, the UDS test was disappointing and gave positive results with <20% of these carcinogens, some of which induced tumours in rat liver and produced DNA adducts in vivo. The TG assay gave positive responses with >50% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus-negative or equivocal carcinogens. This pattern of results was virtually unchanged when the in vitro profile (gene mutagen or clastogen) was taken into account. High sensitivity (ability to detect carcinogens as positive) is only really useful when the specificity (ability to give negative results with non-carcinogens) is also high. Based on small numbers of publications with non-carcinogens, the TG and Comet assays gave negative results with non-carcinogens on 69 and 78% of occasions, respectively. Although further evaluation of the Comet and TG assays, particularly with non-carcinogens, is needed, these data suggest that they both should play a more prominent role in regulatory testing strategies than the UDS test.     

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.     

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay, with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced free radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.     

Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated. Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested. The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage. Results were available from both the alkaline unwinding assay and the mouse lymphoma assay for 61 compounds; they showed a concordance between the 2 assays of 77%. Of the 47 compounds that were positive or equivocal in the alkaline unwinding assay, only carbon tetrachloride and prednisolone were negative in the mouse lymphoma assay, while 12 of the 19 compounds that were negative in the alkaline unwinding assay were positive in the mouse lymphoma assay. These included 3 compounds that interfere with nucleic acid metabolism, and 3 crosslinking agents, which would be expected to produce mutations to a greater extent than strand breaks. The other 6 compounds were anthranilic acid, benzoquinone, p-chloroaniline, diethylmaleate, glucose and procarbazine HCl. Of these only the last is a known carcinogen. It is concluded from the present study that there was good overall agreement between the results of the DNA alkaline unwinding and mouse lymphoma TK locus assays, but that the sensitivity of the alkaline unwinding assay is lower for some classes of compounds. Bearing this in mind, the alkaline unwinding assay is considered suitable as a rapid screen for genotoxic activity in eukaryotic cells.     

The aromatic amine carcinogens o-toluidine and o-anisidine induce free radicals and intrachromosomal recombination in Saccharomyces cerevisiae

Aniline-based aromatic amine carcinogens are poorly detected in short-term mutagenicity assays such as the Salmonella reverse mutation (Ames) assay. More information on the mechanism of toxicity of such Salmonella-negative carcinogens is needed. Aniline and o-toluidine are negative in the Ames assay, but induce deletions (DEL) due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We show here that the DEL assay also detects the genotoxic activity of another aromatic amine carcinogen, o-anisidine, which is also negative in the Salmonella assay. We also show that the DEL assay distinguishes between o-anisidine and its non-carcinogenic structural analog 2, 4-dimethoxyaniline. We have investigated whether the ability of the DEL assay to detect the carcinogens and to distinguish between the carcinogen/non-carcinogen pair is linked to rises in intracellular free radical species following exposure to the carcinogens. Toxicity induced by all three compounds was reduced in the presence of the free radical scavenger and antioxidant N-acetyl cysteine, recombination induced by o-anisidine and o-toluidine was also reduced by N-acetyl cysteine. All three compounds induced oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate. Superoxide dismutase-deficient strains, however, were hypersensitive to cytotoxicity induced by o-toluidine and o-anisidine but not by the non-carcinogen 2,4-dimethoxyaniline, indicating a different potential for generating superoxide radical between the carcinogens and the non-carcinogen analog. The results indicate that the yeast DEL assay is a useful tool for investigating the genotoxic activity of aromatic amine carcinogens.     

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer.     

Microbial mutagenicity of selected hydrazines

Selected hydrazines and related compounds were examined for their mutagenic activity in S. typhimurium strains TA1535 and TA1537. These in vitro assays were conducted with and without metabolic activation by Aroclor-induced rat-liver enzymes. Relatively high levels of mutagenicity were observed with phenylhydrazine X HCl, methylhydrazine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzenediazonium tetrafluoroborate, the stabilized salt of a carcinogenic metabolite of agaritine; only low levels of mutagenicity were observed with other compounds, although most are strong carcinogens. Several of the compounds were highly toxic to the bacteria, and detection of mutagenicity was enhanced by calculating the increase in mutagenic activity on the basis of the surviving fractions of bacteria.     

E,E-2-Benzylidene-6-(nitrobenzylidene)cyclohexanones: syntheses, cytotoxicity and an examination of some of their electronic, steric, and hydrophobic properties

Three series of structurally isomeric 2-benzylidene-6-(nitrobenzylidene) cyclohexanones 1-3 were prepared and evaluated against human Molt/C8 and CEM T-lymphocytes as well as murine L1210 cells. The IC(50) values of the majority of compounds are less than 10microM and in some assays, the figures for 1d and 1e are submicromolar. Correlations were discerned between cytotoxic potencies and the atomic charges on one of the olefinic carbon atoms, the torsion angles between an aryl ring, and the adjacent unsaturated group as well as logP values. Three representative compounds were examined for their effect on respiration in rat liver mitochondria.     

High mutagenic potency of several polycyclic aromatic hydrocarbons induced by liver postmitochondrial fractions from control and xenobiotic-treated immature carp

The metabolism of carcinogens in fish was examined by measuring the activation of different polycyclic aromatic hydrocarbons (PAH) by carp (Cyprinus carpio L.) liver post-mitochondrial fractions (S9) using the Salmonella typhimurium TA100 reverse mutation assay. For this study, 1 non-carcinogen, anthracene (AN), and 4 carcinogens, chrysene (CHR), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC) and 7,12-dimethylbenzanthracene (DMBA), were chosen. The bioactivating potency of the metabolic systems of carp pretreated with phenobarbital (PB), 3MC or Aroclor 1254 (ARO) were compared to uninduced carp liver. The results show that carp liver has the ability to metabolize carcinogenic PAH into mutagenic metabolites, which is enhanced when carp are pretreated with 3MC or ARO, but not with PB. A positive correlation between the induction of aryl hydrocarbon hydroxylase (AHH) activity in carp liver and the mutagenic potencies of CHR, BaP, DMBA and 3MC, has been observed. The bioactivating ability of carp liver S9 was compared with the ability of the same fractions from female Wistar rats (this study) as well as from Sprague-Dawley rats (literature data). When the mutagenic potencies of selected PAH had been normalized on the activity of BaP, the following order of mutagenic activities with S9 fractions from ARO-treated animals was obtained: (1) BaP (1) greater than DMBA (0.26) greater than 3MC (0.22) greater than CHR (0.05) greater than AN (0) for carp; (2) BaP (1) greater than 3MC (0.48) greater than CHR (0.31) greater than DMBA (0.16) greater than AN (0) for Sprague-Dawley rats; and (3) BaP (1) greater than 3MC (0.17) greater than DMBA (0.11) greater than CHR (0) = AN (0) for female Wistar rats. We conclude that carp and rats are very similar in their ability to activate carcinogenic PAH into mutagenic metabolites, which suggests that carp may be very susceptible to the carcinogenic activity of these compounds. According to our results from the mutagenicity study, as well as from the enzyme induction study, we propose the use of carp as a suitable model system for the study of chemical carcinogens.     

Evaluation of the DNA repair host-mediated assay: II. Presence of genotoxic factors in various organs of mice treated with chemotherapeutic agents

The DNA repair host-mediated assay was further calibrated by testing 7 chemotherapeutic agents known to possess carcinogenic activity, namely bleomycin (BLM), cis-diamminedichloroplatinum-II (cis-Pt), cyclophosphamide (CP), diethylstilboestrol (DES), isonicotinic acid hydrazide (isoniazid, INH), natulan (NAT) and mitomycin C (MMC). Differential survival of wild-type and uvrB/recA E. coli strains served as a measure of genotoxic activity. In in vitro assays, BLM, cis-Pt and MMC exhibited high genotoxic activity. The other 4 compounds had no measurable effect on the survival of the two strains, either with or without mouse liver preparations. In the host-mediated assays BLM, cis-Pt, MMC and also NAT induced strong killing of the DNA repair-deficient bacteria recovered from liver, spleen, lungs, kidneys and the blood of treated mice compared to the wild-type strain. The results are not indicative of large organ-specific differences in genotoxically active amounts of the drugs immediately after their application to the host animals. CP, INH and DES did not show geneotix activity in these assays even at very high exposure levels. To compare the genetic endpoint measured in the DNA repair assays, i.e. induction of repairable DNA damage, with the induction of gene mutations, the ability of the 7 drugs to induce valine-resistant (VAL^r) mutants in E. coli was measured in host-mediated assays under identical treatment conditions. INH showed considerable mutagenic activity in E. coli cells recovered from liver and spleen, while BLM and MMC induced a 3–4-fold increase in VAL^r mutants above spontaneous levels. The other compounds showed no mutagenic activity under these in vivo conditions. From these results it can be concluded that (i) the type of primary DNA lesions produced by these chemotherapeutic agents (cross-links by MMC and cis-Pt, and strand breaks by BLM and possibly by NAT; base alkylation by INH) appears to determine whether a compound will be highly positive in the DNA repair assay as in the case of BLM, cis-Pt, MMC and NAT, and less effective in inducing mutations under similar conditions, or whether the opposite will occur, as in the case of INH; (ii) DES and CP probably do not interact sufficiently with bacterial DNA to show an effect in either of the genetic endpoints; and (iii) the present DNA repair host-mediated assay may represent a sensitive, rapid and economic method for monitoring genotoxic factors in various organs of experimental animals which have been treated with cytostatic drugs.     

Continuous spectrophotometric assays for cytosolic epoxide hydrolase

Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase are described. The assays are based on the differences in the ultraviolet spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) is accompanied by a decrease in absorbance at 302 nm, while the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produces an increase in absorbance at 315.5 nm. The Km, Vmax values for ENP5 and EQU5 with purified mouse liver cytosolic epoxide hydrolase were 1.7 microM, 11,700 nmol/min/mg and 25 microM, 8300 nmol/min/mg, respectively. Both substrates are hydrolyzed significantly faster than trans-stilbene oxide, which is currently the most commonly used substrate for measuring cytosolic epoxide hydrolase activity. No spontaneous hydrolysis of the substrates is detectable under normal assay conditions. The assays are applicable to whole tissue homogenates as well as purified enzyme preparations. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examined and found to be very poor substrates for cytosolic epoxide hydrolase from mouse liver.     

Consideration of both genotoxic and nongenotoxic mechanisms in predicting carcinogenic potential

Bacterial and cell culture genotoxicity assays have proven to be valuable in the identification of DNA reactive carcinogens because mutational events that alter the activity or expression of growth control genes are a key step in carcinogenesis. The addition of metabolizing enzymes to these assays have expanded the ability to identify agents that require metabolic activation. However, chemical carcinogenesis is a complex process dependent on toxicokinetics and involving at least steps of initiation, promotion and progression. Identification of those carcinogens that are activated in a manner unique to the whole animal, such as 2,6-dinitrotoluene, require in vivo genotoxicity assays. There are many different classes of non-DNA reactive carcinogens ranging from the potent promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that acts through a specific receptor, to compounds that alter growth control, such as phenobarbital. Many compounds, such as saccharin, appear to exhibit initiating, promotional and/or carcinogenic activity as events secondary to induced cytotoxicity and cell proliferation seen only at the chronic lifetime maximum tolerated doses mandated in rodent bioassays. Simple plus/minus vs. carcinogen/noncarcinogen comparisons used to validate the predictivity of bacterial and cell culture genotoxicity assays have revealed that a more comprehensive analysis will be required to account for the carcinogenicity of so many diverse chemical agents. Predictive assays and risk assessments for the numerous types of nongenotoxic carcinogens will require understanding of their mechanism of action, reasons for target organ and species specificity, and the quantitative dose-response relationships between endpoints such as induced cell proliferation and carcinogenic potential.