Response of the ke test to NCI/NTP-screened chemicals. II. Genotoxic carcinogens and non-genotoxic non-carcinogens.

A physico-chemical carcinogen-screening test was used to measure the rate constants of electron attachment, kes, of 105 chemicals that had been screened in long-term rodent bioassays and short-term in vitro tests by the NCI/NTP. In the ke test, a pulse-conductivity technique is used to generate and monitor the decay of excess electrons that serve as nucleophilic surrogates for the target tissue of rodents. Of the 61 chemicals that had been found to be rodent carcinogens as well as Salmonella mutagens, 36 yield kes that are equal to or greater than the diffusion-controlled ke of carbon tetrachloride and are considered to be positive ke test responses. In contrast, 29 of the remaining 44 chemicals that are putative non-carcinogens and non-mutagens yield kes that are negative ke test responses. These results are combined with the ke responses of 46 non-mutagenic carcinogens and 20 mutagenic non-carcinogens that were reported earlier and are evaluated to determine the degree to which the measure of electron-accepting capacity that ke provides complements or overlaps the electrophilicity or DNA reactivity of chemicals that is indicated by positive mutagenicity responses in the Ames Salmonella tester strains or by positive structural alerts, S/As, of the chemicals. The combined ke test results indicate that the overall predictivity of the ke test is comparable to and complements the Ames Salmonella test and S/As in identifying rodent carcinogens. Moreover, the electrons serve as non-discriminate nucleophilic targets for both genotoxic and non-genotoxic electron-accepting molecules and appear to attach with equal efficiency to carcinogens that are active in various tissues of rodents. This property of excess electrons suggests that the predictivity of the ke test could be enhanced by combining the measured ke with an appropriate lipophilicity or pharmacokinetic parameter. A pre-chemical electron-transfer step that had been proposed to precede chemical interactions between the carcinogen and target tissue is discussed in light of recent developments in electron-donor/-acceptor chemistry and in the application of structure--activity relationships to identify carcinogens.     

Response of the ke test to NCI/NTP-screened chemicals. III. Complementary value of ke in screening for carcinogens.

The value of using a physico-chemical carcinogen-screening test, the ke test, in conjunction with the Salmonella typhimurium/microsome assay (the Ames test) and/or structural alerts of reactivity (the S/A test), is analyzed on the basis of the response of the three tests to 171 chemicals of known rodent carcinogenicity. The Ames test is widely used to screen chemicals for potential carcinogenicity; however, its relatively low sensitivity (proportion of true positives among carcinogens tested) has prompted a search for complementary tests that increase sensitivity without an unacceptable decrease in specificity (proportion of true negatives among non-carcinogens tested). The S/A test is a structural analysis based on recognition of chemicals groups likely to react with DNA. The S/A test does not complement the Ames test well, because of the high similarity of responses (dependence) between these two tests. The ke test measures the affinity of a test chemical for electrons, and has a sensitivity and specificity comparable to the Ames test. The ke test is shown in this work to complement both the Ames test and the S/A test. Addition of the ke test to either the Ames test or the S/A test results in a substantial decrease in false negatives and an approximately equal increase in false positives, which is a trade-off that would be desirable in all but the least risk averse situations. The S/A and ke battery has a sensitivity of > 0.9, and could be applied to untested chemicals without any biological testing. In view of these observations, it is proposed that the ke test be considered in developing future strategies to optimize the screening of potential carcinogens in the most cost-effective manner.     

Effects of SO2 or NOx on toxic and genotoxic properties of chemical carcinogens. I. In vitro studies

This paper describes in vitro studies on the effects of environmentalpollutants (SO₂ /NO_x) in biological systems. Basic physical,chemical and biochemical parameters were analyzed to establishthe rate of (SO₂ /NO_x) absorption by the culture medium. Itwas shown that the pH remains constant for 24 h of exposureto gas concentrations up to 50 p.p.m. The concentration of ionsresulting from absorption of each pollutant in the liquid phaseis dependent on their concentration in the gas phase and onexposure time. Short exposure times and high gas dosages resultedin similar doses in the medium as long exposure periods andlow gas dosages. The activities of a human serum standard (alkalinephosphatase, ALP; aspartate amino transferase, AST; alanineamino transferase, ALT; -glutamyltransferase, -GT; lactate dehydrogenase,LDH) were determined after gaseous exposure to SO₂ and NO_x.The results revealed a distinct decrease in the activity ofLDH after 1,3 and 5 h exposure to 200 p.p.m. SO2. The effectsof the pollutants were assayed in vitro using fetal hamsterlung cells (FHLC), rat hepatocytes and the cell line CO60. Forthe determination of toxic effects, it was shown that the platingefficiency was a more sensitive parameter than the assay fortrypan blue exclusion. Toxicity indicated as an increase ofLDH leakage was not observed from FHLC in culture. Instead,a decrease of LDH was found following SO2 exposition. This decreasewas similar to that observed for the human serum standard. Theinduction of DNA singlestrand breaks was determined as a measureof genotoxic effects. SO₂ application decreased the rate ofDNA singlestrand breaks induced by N-nitroso-acetoxymethylmethylaminein both FHLC and in rat hepatocytes. SO₂ or NO, treatment ofCO60 cells for 1 h did not result in the induction of DNA amplification.HSO₃ ^- added directly to the medium as the sodium salt, however,distinctly induced the amplification of SV40 DNA. The amplificationrates induced by benzo[a]pyrene or dimethylbenzanthracene wereneither influenced by SO₂, NO_x nor HSO₃^-. An additive effectof HSO₃^- with either benzo[a]pyrene or dimethylbenzanthracenefor this biological parameter was therefore not observed.     

A new approach to identifying genotoxic carcinogens: p53 induction as an indicator of genotoxic damage

The tumor suppressor gene p53 encodes a nuclear phosphoprotein which is critical for cell cycle control and prevention of uncontrolled cell proliferation that can lead to cancer. Previous studies have shown that cells respond to DNA damage by increasing their levels of p53, which then acts to prevent replication of damaged DNA. This study examined the effects on p53 protein levels of several different categories of chemical carcinogens. N-Methyl-N'-nitro-nitrosoguanidine and N-ethyl-N- nitrosourea, two direct-acting genotoxic (DNA-reactive) carcinogens, caused p53 induction as early as 2 h following treatment, with peak increases within 4-12 h. Aflatoxin B1 and 2-acetylaminofluorene, indirect-acting genotoxic carcinogens, caused a later induction of p53, with the peak increase appearing between 16 and 24 h following treatment. These observations demonstrate a correlation between p53 induction pattern and DNA damaging mechanism of genotoxins. Phenol, diethylstilbestrol and ethylacrylate also induced increases in cellular p53. The half-life of p53 protein was increased in cells treated with genotoxic agents. On the other hand, the epigenetic (non-DNA-reactive) carcinogens azathioprine and saccharin, as well as two substances generally considered to be non-carcinogens, dimethylsulfoxide and benzethonium chloride, had no effect on p53 protein levels of treated cells. Measurement of the cytotoxic effects of each of these chemicals led to the conclusion that p53 protein induction is not a general, non- specific consequence of the cytotoxic effect of these genotoxins. These results suggest that measurement of p53 protein induction may be an effective tool to identify environmental genotoxins.      

Nine-week detection of six genotoxic lung carcinogens using the rasH2/BHT mouse model

A 9-week in vivo rasH2/butylhydroxytoluene (BHT) model for the detection of genotoxic lung carcinogens was validated, using six potent positive test compounds, dimethylnitrosamine (DMN; 15 mg/kg, i.p.), diethylnitrosamine (DEN; 100 mg/kg, i.p.), ethylnitrosourea (ENU; 120 mg/kg, i.p.), 3-methylcholanthrene (MC; 100 mg/kg, i.p.), 7,12-dimethylbenz(a)anthracene (DMBA; 5 mg/kg, i.g.) and benzo(a)pyrene (B(a)P; 80 mg/kg, i.p.), each given to rasH2 mice of both genders by single administration for initiation followed by promoter BHT treatment. Statistically significant increase in the incidence and multiplicity of lung tumors was observed in rasH2 mice treated with BHT following exposure to all of the carcinogens tested. The data overall suggest the rasH2/BHT model to be a powerful screening tool for genotoxic lung carcinogens.     

The colonic response to genotoxic carcinogens in the rat: regulation by dietary fibre

The apoptotic response to DNA damage appears to be an innate biological mechanism for protection against tumourigenesis. It is possible that agents that protect against colorectal cancer act by enhancing the apoptotic deletion of cells suffering DNA damage, with consequent removal of those with tumourigenic mutations. We examined the acute apoptotic response to genotoxic carcinogens ("AARGC") in colonic epithelium and the possibility that dietary fibres of different fermentability might regulate AARGC. To fully define the time-course and nature of AARGC in response to the carcinogen azoxymethane (AOM), a single injection of AOM (10 mg/kg) was given to rats and apoptosis monitored in the colon by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling staining over a 72 h period. Having defined the site and time of maximum response, two groups of eight rats were fed diets containing 10% wheat bran fibre (WB; fermentable) or 10% methylcellulose (MC; poorly fermentable) for 4 weeks. Colonic AARGC was compared by light microscopy; lumenal short chain fatty acids (SCFAs) and pH were measured as indicators of the fermentative environment. AOM-induced AARGC was maximal at 8 h and greater in distal compared with proximal colon. Apoptotic cells were situated predominantly in the lower half of the crypt, with the median at position 9 indicating involvement of daughter as well as stem cells. There was no "second wave" of apoptosis within 72 h as follows irradiation in small intestine. Distal colonic AARGC in rats fed WB was twice that in rats fed MC (P < 0.01). Compared with MC, WB significantly lowered lumenal pH and increased all SCFAs including butyrate, while proliferation did not differ between the fibres. Certainly, dietary fibres can regulate AARGC and further studies are warranted to determine if this biological effect is the way in which dietary factors regulate tumourigenesis. Lumenal generation of butyrate may enhance AARGC as butyrate is proapoptotic in vitro.     

Failure of genotoxic carcinogens to produce tumors in human skin xenografts transplanted to SCID mice

Chemical carcinogenesis of human skin was investigated usinghuman skin xenografts (16 full thickness and 48 split thicknessskin grafts) transplanted to CB-17-scid (SCID) mice. Topicalapplication of a carcinogen, i.e. 7, 12-dimethyl-benz[a]anthracene(DMBA), benzo[a]pyrene, methylchol-anthrene or N-methyl-N'-nitro-N-nitrosoguanidine,to the human skin xenografts once a week for 25–30 weeksfailed to produce skin tumors. Both DMBA application plus UV-Birradiation and alternate applications of the above four carcinogensin combination with UV-B irradiation also failed to producetumors. All of these treatments induced skin papillomas in skinsof host SCID mice. DMBA induced skin papillomas in allogenicCD-1 mouse skin grafts transplanted to SCID mice. These resultsindicate that susceptibility of human skin to these carcinogenicstimuli is much lower than that of mouse skin.     

32P-postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents

Carcinogen - DNA adducts were detected and determined by ³²P-postlabelingassay after exposure of mouse or rat tissues in vivo to a totalof 28 compounds comprising 7 arylamines and derivatives, 3 azocompounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons,and 4 methylating agents. DNA was isolated from mouse skin,mouse liver, and rat liver after treatment with the individualcarcinogens, then digested enzymatically to deoxyribonucleoside3'-monophosphates, which were converted to 5'-³²P-labeled deoxyribonucleoside3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [³²P]phosphatetransfer from [-³²P]ATP. The nucleotides were resolved by anion-exchanget.l.c. on polyethyleneimine-cellulose and detected by autoradiography.The determination of low levels of DNA binding of the aromaticcarcinogens entailed the removal of normal nucleotides priorto the resolution of adduct nucleotides. For this purpose, analternative procedure employing reversed-phase t.l.c. was devisedwhich offered advantages for the detection of quantitativelyminor adducts. The procedures described enabled the detectionof 1 aromatic DNA adduct in 10⁸ normal nucleotides, while thelimit of detection of methylated adducts was 1 adduct in 6 x10⁵ nucleotides. The results show that a great number of carcinogen-DNAadducts of diverse structure are substrates for ³²P-labelingby polynucleotide kinase-catalyzed phosphorylation. Becausecovalent DNA adduct formation in vivo appears to be an essentialproperty of the majority of chemical carcinogens, ³²P-postlabelinganalysis of carcinogen -DNA adducts in mammalian tissues mayserve as a test for the screening of chemicals for potentialcarcinogenicity.     

Detection of mutagenic impurities in carcinogens and noncarcinogens by high-pressure liquid chromatography and the Salmonella/microsome test.

We have used high-pressure liquid chromatography and the Salmonella/microsome mutagenicity test to look for mutagenic impurities in 11 carcinogens and noncarcinogens. Because of the million-fold range in mutagenic potency observed in the Salmonella test, even trace amounts of potent mutagenic impurities in a nonmutagenic compound could be detected. The mutagenicity of 7-hydroxy-2-acetylaminofluorene, a noncarcinogen in the standard animal carcinogenicity tests, is shown to be due to a small amount of impurity, which is probably the potent carcinogen 2-acetylaminofluorene. This is discussed in relation to the statistical limitations of animal carcinogenicity tests. We also discuss the role of mitogenic impurities in assessing the mutagenicity of environmental (and industrial) chemicals with high-sensitivity mutagenicity assays, such as the Salmonella/microsome test.     

Evaluation of the transgenic p53+/- mouse for detecting genotoxic liver carcinogens in a short-term bioassay.

The transgenic p53-deficient heterozygous (p53+/-) mouse is prone to both spontaneous and induced tumors and has been proposed for use in a sensitive, short-term (6 months) assay for identifying genotoxic, multispecies carcinogens. It is not clear, however, if a short-term assay with p53+/- mice detects agents that target certain organs, in particular, the liver. In this study, we treated neonatal male p53+/- and p53+/+ mice with the genotoxic carcinogens dimethylnitrosamine (DMN), 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), and 6-nitrochrysene (6-NC). In keeping with the methodology of the proposed short-term assay, the p53+/- mice were evaluated for tumors 7 months after treatment. Wild-type neonatal mice treated with genotoxic carcinogens are known to develop tumors within 1 year; hence, the p53+/+ animals used as controls were subjected to pathological examination at 1 year of age. Our results showed that PhIP was not tumorigenic in either group of mice. Liver tumor incidence increased significantly in the p53+/+ mice treated with DMN and 6-NC, indicating that the conditions of the bioassay were conducive to the promotion of liver tumorigenesis. On the other hand, these two chemicals failed to induce a significant increase in liver tumors in the p53+/- mice by seven months. This result suggests that a deficiency in the amount of p53 protein does not lead to accelerated liver tumorigenesis in mice, and contrasts with previous reports that show a decreased latency of tumors in non-liver targets.     

Detection of mammalian carcinogens with an immunological DNA synthesis-inhibition test.

There is a close relationship between genotoxicity, mutagenicity and carcinogenicity. But the controversy of which short-term test system best recognizes human carcinogens is still going on. Currently, the Salmonella gene mutation assay ('Ames test') is the most widely used test for the screening of mutagens. However, many in vitro tests hold unsatisfactory validity data, presumably because of the inability of present short-term tests to detect non-genotoxic carcinogens, which are increasingly being brought into focus in the discussions of genesis of cancer. One principle often neglected in this context is the property of genotoxic agents to inhibit replicative DNA synthesis in (proliferating) eukaryotic cells. We believe that this early response to DNA damage is important in the multistage process of carcinogenesis. Accordingly, we proposed that a DNA synthesis-inhibition test should be included in the test batteries for carcinogen screening. In this paper we report the development of an appropriate DNA synthesis-inhibition test based on immunological techniques.     

Re: Yang, J. and Duerksen-Hughes, P. (1998) A new approach to identifying genotoxic carcinogens: p53 induction as an indicator of genotoxic damage. Carcinogenesis, 19, 11171125.

Dear Sir, In a recent issue of the Journal, Yang and Duerksen-Hughes presenta new in vitro approach for identifying genotoxic carcinogens.In this study they used p53 induction in vitro as an indicatorof genotoxic damage. The authors suggest that this endpointcould be used as an effective tool for identifying environmentalcarcinogens. In our opinion, p53 induction has clear weaknesses as an endpointfor in vitro genotoxicity testing. It is induced by many types     

Effects of SO2 or NOx on toxic and genotoxic properties of chemical carcinogens. II. Short term In vitro studies

Short term in vivo studies were performed to study biologicaleffects of the common air pollutants SO₂ or NO_x and their influenceon the genotoxic activities of nitrosamines. Hepatocytes andlung cells were isolated from Sprague-Dawley rats which hadinhaled 50 p.p.m. of SO₂ or NO_x for 2 weeks. After incubatingthe cells for 1 h, genotoxicity was determined in hepatocytesby measuring DNA single-strand breaks induced by iV-nitroso-acetoxymethylmethylamine,N-nitrosodimethylamine and N-nitrosomethylbenzylamine. Parametersof toxicity (trypan blue exclusion and leakage of serum enzymes)were determined in both liver and lung cells also following1 h incubation. The activities of aryl hydrocarbon hydroxylase(AHH), nitrosodimethylamine demethylase (NDMA-D) and glutathione-S-transferase(GST) were determined in subcellular microsomal fractions isolatedfrom lung and liver tissues. Finally, as a measure of overalltoxicity, the activities of various serum enzymes were determinedin the blood serum of the rats. It was found that the inductionof DNA single-strand breaks by three nitrosamines was decreasedin hepatocytes from SO₂-treated animals. The viability of rathepatocytes and of rat lung cells, as determined by trypan blueexclusion, was similar in all three treatment groups immediatelyafter isolation, as well as after 1 h incubation with DMSO orwith the nitrosamines. In contrast, the leakage of enzymes wasdifferent in hepatocytes of SO₂-treated rats, since lactatedehydrogenase activity was decreased. Leakage of enzymes fromthe lung cells did not differ from group to group, but was lowerthan from hepatocytes. Foreign compound metabolizing enzymeswere mainly decreased in NO_x-treated animals, namely AHH, NDMA-Dand GST in liver and GST in the lung. For SO₂-treated animalsNDMAD was increased in liver and GST was decreased in lung.Blood serum enzyme levels were not greatly different from eachother, except for lactate dehydrogenase which was elevated inSO₂-exposed animals.     

Response of experimental animals to human carcinogens: an analysis based upon the IARC Monographs programme

Only the results of epidemiological studies can be used to establisha causal relationship between an exposure to an agent and humancancer; however, such studies often cannot be carried out dueto limitations of population or latent period or to the presenceof mixed exposures. It is essential, therefore, that the validitybe established of extrapolating to humans the results obtainedfrom long-term carcinogenicity tests in animals. The responsesof experimental animals to known and suspected human carcinogens,as evaluated in the IARC Monographs series, were analysed asan indication of the sensitivity of animal tests for predictinghuman carcinogens. Although the response was high - 84% - itwould have been even higher had all the compounds been adequatelytested experimentally. An additional finding was that for manyexposures causally related to human cancer, there is a targetorgan in common between humans and at least one animal species,despite many inherent physiological differences. These findingsshow clearly the importance of experimental carcinogenicitystudies in the primary prevention of cancer.     

Carcinogenic chemicals enhance mouse leukemia virus infection in contact-inhibited culture: a new simple method of screening carcinogens.

Carcinogenic chemicals enhanced infection in contact-inhibited cells with mouse leukemia virus. A correlation was found between the enhancing effects and the carcinogenicities of the 40 chemicals tested. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate did not enhance viral infection.     

Feasibility of monitoring populations to detect environmental carcinogens.

In relation to cancer, monitoring, i.e. "continuing observation in order to decide when changes in incidence or mortality may have occurred" can be of two types: (a) passive (or routine) monitoring using routinely collected cancer statistics for populations in general (b) active (or exposure-oriented) monitoring in which the cancer experience of groups with specific exposures is compared with that of the non-exposed. The advantages and disadvantages of mortality and morbidity data for passive monitoring are listed. It is concluded that passive monitoring can rarely do more than indicate areas for further study. The potential of active monitoring is examined by consideration of industrial and occupational exposures, the follow-up of persons on prolonged drug therapy (including transplacental effects) and changes in cancer risk following migration. As such studies are often univariate, it may not be possible to allow for the effect of other factors, and confirmatory studies will often be required. It is belived that priority should currently be given to monitoring occupational exposures, particularly those involving chemicals shown by animals studies to have carcinogenic activity. To monitor everybody for everything will result in nothing. The social responsibilities which devolve on monitors, the authorities, both sides of industry and the general public as a result of the establishment of monitoring systems are discussed. It is concluded that monitoring, active or passive, does not represent a quick road to the identification of environmental carcinogens but can point to products or processes likely to have a cancer risk - a risk which would have to be confirmed or otherwise by other methods.