Screening assays for carcinogenic agents and mixtures: an appraisal based on data in the IARC Monograph series

To determine whether genotoxic and non-genotoxic carcinogens contribute similarly to the cancer burden in humans and which types of short-term test are more relevant for predicting human hazards, an analysis was performed on agents that were evaluated in IARC Monographs Supplements 6 and 7 for their carcinogenic effects in humans and animals and for activity in short-term genotoxicity tests. The prevalence of genotoxicity among four groups of agents, consisting of established human carcinogens (group 1, n = 30), probable human carcinogens (group 2A, n = 37), possible human carcinogens (group 2B, n = 113) and agents with limited evidence of carcinogenicity in animals (a subset of group 3, n = 66) was determined. Each of the groups 1, 2A and 2B contained a high proportion (80-90%) of genotoxic carcinogens, which were also multi-species or multi-tissue carcinogens. The distribution of carcinogenic potency in rodents did not reveal any specific characteristic of the human carcinogens in group 1 that would differentiate them from agents in groups 2A, 2B and many in group 3. Although limited by the data-base available through the Monographs series, this analysis implies that genotoxic carcinogens add more to the human cancer burden than non-genotoxic carcinogens. Thus, the continued use of in vitro/in vivo short-term tests, involving as endpoints DNA chromosomal or mutational damage, to identify genotoxic carcinogens or in the isolation of carcinogenic components in complex mixtures is fully justified. It is concluded that (a) an agent or complex mixture with unknown carcinogenic potential showing sufficient evidence of activity in genotoxicity assays in vitro or in vivo is likely to represent a hazard to humans and (b) an agent or complex mixture showing lack of activity in this spectrum of genotoxicity assays should undergo evaluation for carcinogenicity for rodent bioassay, in view of the present lack of validated short-term tests for non-genotoxic carcinogens.     

Identification of trihalomethanes in drinking water and assessment of their toxicity, mutagenicity and carcinogenicity

Dichlorobromomethane (DCMB) and dibromochloromethane (DBCM) isolated from chlorinated drinking water were tested for toxicity, mutagenicity and carcinogenicity. Both agents proved mutagenic in a "dessicator" modification of the Ames test using Salmonella typhimurium TA98 and TA100 in the presence of exogenous metabolic activation. In aquarium Danio rerio fish tests, LD50/30 was 250 mg/l for both compounds. Both agents induced hepatocellular carcinoma in fish: DCMB--in 11 out of 29 animals (at 16.5 weeks) and DBCM--in 3 out of 16 (at 26.5 weeks). These data merit further investigation of the agents' carcinogenicity in chronic experiments in rodents.     

Methodological approaches to the study of the carcinogenic properties of substances

Since the methods of investigation of carcinogenicity of different agents have changed drastically, relevant manuals need to be revised and supplemented. A new concept of carcinogenic agents evaluation, criteria for their selection and study are discussed. Working out of criteria for establishing priorities of hazardous substance selection is of vital importance.     

Evaluation of the carcinogenic risk of environmental chemicals

Many carcinogens have been detected in our environment, and chemicals which have been shown to induce cancers in experimental animals have been largely eliminated. However, there are still many chemicals which have not been studied in rodent carcinogenicity tests. In this paper, we review the evaluation of carcinogenic risk of environmental chemicals to humans. Carcinogenic risk to humans is evaluated primarily in three different phases: carcinogenicity studies in animal experiments, in vitro short-term screening studies, and finally human epidemiological studies. There are 30 chemicals, processes or industries which have been subject to these three phases and determined to be carcinogenic to humans by the International Agency for Research on Cancer (IARC). Carcinogenic risk of chemicals should be evaluated quantitatively by their threshold doses, TD50 and virtually safe dose (VSD). Furthermore, the target organ of the carcinogen should also be taken into account, which has been emphasized by the demonstration of carcinogenicity of BHA in the forestomach of rats. Summational, synergistic, antagonistic and inhibitory effects of chemicals should also be discussed. It is important to establish a guideline for detecting and eliminating environmental carcinogens in order to prevent cancer in humans.     

Prediction of carcinogenic potential by a toxicogenomic approach using rat hepatoma cells

The long-term rodent bioassay is the standard method to predict the carcinogenic hazard of chemicals for humans. However, this assay is costly, and the results take at least two years to produce. In the present study, we conducted gene expression profiling of cultured cells exposed to carcinogenic chemicals with the aim of providing a basis for rapid and reliable prediction of carcinogenicity using microarray technology. We selected 39 chemicals, including 17 rat hepatocarcinogens and eight compounds demonstrating carcinogenicity in organs other than the liver. The remaining 14 were non-carcinogens. When rat hepatoma cells (MH1C1) were treated with the chemicals for 3 days at a non-toxic dose, analysis of gene expression changes with our in-house microarray allowed a set of genes to be identified differentiating hepatocarcinogens from non-carcinogens, and all carcinogens from non-carcinogens, by statistical methods. Moreover, optimization of the two gene sets for classification with an SVM and LOO-CV resulted in selection of 39 genes. The highest predictivity was achieved with 207 genes for differentiation between non-hepatocarcinogens and non-carcinogens. The overlap between the two selected gene sets encompassed 26 genes. This gene set contained significant genes for prediction of carcinogenicity, with a concordance of 84.6% by LOO-CV SVM. Using nine external samples, correct prediction of carcinogenicity by SVM was 88.9%. These results indicate that short-term bioassay systems for carcinogenicity using gene expression profiling in hepatoma cells have great promise.     

Lack of correlation between alkaline DNA fragmentation and DNA covalent binding induced by polychloroethanes after in vivo administration. Problems related to the assessment of a carcinogenic hazard

The DNA-damaging activity of polychloroethanes was tested in mouse liver by the fluorometric assay of DNA unwinding. With the exception of 1,2-dichloroethane, all components of this chemical class had negative results. The failure of the parameter alkaline "DNA fragmentation" to detect the DNA-damaging activity of polychloroethanes is in sharp contrast with the measurement of DNA covalent binding, another short-term parameter of genotoxicity. Since covalent DNA adducts appear to be quantitatively well correlated with the oncogenic potencies of chloroethanes in liver, the negative results obtained with the present method can perhaps be explained in terms of quality of DNA adducts; these may be incapable of producing DNA breaks or alkali-labile sites detectable as alkaline DNA fragmentation. It is however worth noting that carcinogenicity of chloroethanes appears to depend not only on DNA damaging capability, but also on promoting activity during the carcinogenic process.     

Evaluation of potential carcinogenic hazards of lead and lead compounds

A considerable percentage of Russian population, as well as in other countries, are at risk of exposure to lead as an industrial highly toxic hazard. It is notorious for polytropic influence, high stability both in human body and environment; it has a cumulative effect and a possible distant after-effect. An IARC working group carried out an evaluation of the data on lead carcinogenicity in industrial cohorts and found no suspicious risks. Inorganic lead compounds were classified as "probably carcinogenic to humans" (group 2B). Later on, the IARC Working Group (2004) referred those substances to group 2A (carcinogenic to humans).     

Tumor initiating activity of Helicobacter pylori water extract on mouse skin carcinogenesis

Helicobacter pylori (H. pylori) infection has been associated with gastric carcinogenesis, but responsible and detail mechanisms are insufficient by the absence of adequate data. To obtain direct evidence regarding the carcinogenicity of H. pylori, we investigated the initiating and promoting activity of H. pylori water extract (HPE) in two-stage mouse skin carcinogenesis model. HPE treatment, as an initiation, significantly enhanced tumor formation compared with control group. Moreover, HPE treatment increased production of 8-hydroxydeoxyguanosine in epidermal cells and HPE-initiated/TPA-promoted papillomas demonstrated a point mutation of the Ha-ras gene. These results suggest an initiating activity of HPE on two-stage mouse skin carcinogenesis.     

Use of the data of quantitative analysis of organo-systemic distribution of tumors in the evaluation of real hazards of chemical carcinogens

The study was concerned with objective criteria for identification of actual carcinogenic hazards. It used quantitative analysis data on morphologic structures of tumors induced in 12565 albino noninbred rats in the course of carcinogenicity evaluation of 112 chemical agents. The totality of all the data were analyzed versus morphologic parameters of tumors and carcinogenicity of the agents. Spontaneous tumors turned to be the cause of death in animals treated with noncarcinogenic or slightly carcinogenic agents whereas a completely different spectrum of tumors were responsible for lethality in those receiving potent carcinogens. It was inferred that increased incidence of spontaneous tumors following treatment with maximum tolerable doses can not be used as a criterion of actual blastogenicity of an agent.     

The role of the stages of initiation and promotion in phenotypic diversity during hepatocarcinogenesis in the rat.

Differences in the distribution of phenotypic alterations in initiated and promoted cell populations reflect both the dose and the action of the specific initiating agent, as well as the mechanism of action of the promoting agent. The use of multiple phenotypic markers to characterize AHF should allow further experimentation on the characteristics of promoting agents in hepatocarcinogenesis, especially their ability to promote separate populations of initiated cells, and on those characteristics of initiated cells that enable certain ones to survive and multiply in the specific environment provided by a promoting agent. Studies of promoting agents with differing mechanisms of action should further allow questions of reversibility, substitution and additivity of the actions of promoting agents to be addressed. The possibility that individual promoting agents do not enhance the growth of the entire population of initiated cells indicates that study of combinations of promoting agents is an important future direction of research. Therefore, information on the characteristics of promoting agents, singly and in combination, is necessary to assess more accurately the contribution of promoting agents to the carcinogenic risk for humans distinct from the effects of initiating agents and complete carcinogens.     

Quantitative relationship between mutagenic potency in the Ara test of Salmonella typhimurium and carcinogenic potency in rodents. A study of 11 direct-acting monofunctional alkylating agents

This work attempted to derive a quantitative relationship between mutagenicity and carcinogenicity by examining the association between mutagenic potency in the Ara test of Salmonella typhimurium and carcinogenic potency in rodents. Mutagenesis was monitored by selecting forward mutations to L-arabinose resistance. Lethality was measured at equivalent experimental conditions to those of mutant yield by using a mixed population of a pair of isogenic strains distinguished by their differential nutritional requirements. The study was carried out with a group of 11 direct-acting monofunctional alkylating agents, which failed to show any quantitative correlation in the histidine reverse-mutation test. Our data suggest that the mutagenic efficiency of the compounds is directly proportional to the magnitude of the maximum yield of L-arabinose resistance mutants and inversely proportional to the dose and the number of lethal hits at which the maximum yield occurs. A highly significant correlation (r10 = 0.86, P less than 0.01) was found between the mutagenic efficiencies of the compounds in the Ara test and their carcinogenic potencies in rodents, expressed as TD50 ('tumor dose' 50) values. The result suggests that the Ara forward-mutation test of S. typhimurium might be capable of reflecting the relative potency of animal carcinogens, at least when confined to particular chemical classes. A more generic and definitive conclusion about the predictive value of the Ara test would require this analysis to be extended to other types of genotoxic carcinogens.     

Inhaled particles and lung cancer, part B: paradigms and risk assessment

Poorly soluble particles of low toxicity (PSP), such as CB, TiO(2) and coal mine dust, have been demonstrated to cause lung cancer in rodents, being most pronounced in rats. Adequate epidemiologic studies do not clearly indicate increased lung cancer rates in humans exposed to such particles. This has caused controversial positions in regulatory decisions on PSP on different levels. The present review discusses the current paradigms in rodent particle carcinogenicity, i.e., (i) role of particle overload and of persistent inflammation and (ii) fibrosis as an intermediate step in particle-induced lung cancer with regard to human risk assessment. Fibrosis, which is usually considered a precursor of lung cancer in humans, was not related to lung tumors in an animal study using 6 different particles, each at 3 dosages. Lung tumors after both inhalation and intratracheal instillation of PSP are related to particle surface dose, which forwards hazard assessment at surface-based nonoverload concentrations and a standard setting using surface as an exposure metric. The scarce data available on humans do not support the overload concept but suggest a role for persistent lung inflammation. Differences in antioxidant protection between different rodent species correlate with susceptibility to PSP-induced carcinogenicity and support the need for detailed studies on antioxidant response in humans. Apart from such bridging studies, further focus is also needed on surface chemistry and modifications in relation to their adverse biologic effects.     

The impact of toxicity on carcinogenicity studies: implications for risk assessment

This paper explores the inter-relationship between toxicity,genotoxicity, and carcinogenicity in laboratory rodents. Toour knowledge this is the first attempt to integrate these factorsand evaluate their implications for the process of risk assessment.The evaluation is based on information obtained from 2-yearlaboratory-animal studies involving 99 chemicals. The data suggestthat only seven of the 53 positive carcinogenicity studies exhibitedthe types of target organ toxicity that could have been thecause of all observed carcinogenic effects. Furthermore, noapparent difference in mutagenicity as measured by the AmesSalmonella assay was observed between ‘high dose only’carcinogens and the entire set of carcinogens. These findingssuggest that the number of chemical carcinogens that we canidentify solely through rodent studies as being potential tumorinducers through some indirect mechanism is small. Generallyspeaking, the identification of histopathological effects isnot sufficient in itself for justifying mechanistic assumptions,and supplemental biological information will be necessary toreach definitive conclusions.     

Simian virus 40 and malignant mesothelioma (Review)

Simian virus 40 (SV40) was recognized as a contaminant of early poliovirus vaccines that were provided to millions of individuals in Europe and in the USA between 1955 and 1963. SV40, a DNA virus of the family of papovaviridae, was proven to be oncogenic in rodents and able to transform human and animal cells in vitro. In 1993 SV40 was accidentally discovered to produce mesotheliomas in hamsters when it was injected in visceral cavities. Afterwards, SV40 DNA sequences were detected with significative frequency in human pleural mesotheliomas by using polymerase chain reaction (PCR) and then SV40 DNA oncogenicity was associated with its large T antigen (Tag). This finding was confirmed by many laboratories, while a few research groups failed to replicate these data and argued that the SV40 DNA detection might be a PCR contamination artefact. In this review the dispute is examined in the light of recent experiments performed to identify molecular and cellular aspects of carcinogenicity and/or co-carcinogenicity of SV40 in human mesothelioma.     

Nucleophilic selectivity of alkylating agents and their hypermutability in Drosophila as predictors of carcinogenic potency in rodents

The nucleophilic selectivity (Swain-Scott s constant or initial 7-alkylguanine/O6-alkylguanine ratio in DNA) of 60 alkylating agents, mostly monofunctional or cross-linking was compared to their carcinogenic potency in rodents (median TD50 estimates) and to two genotoxicity indices in Drosophila: (i) hypermutability, measured by the increased frequency of induced sex-linked recessive lethal mutations (SLRL) in a strain defective in DNA excision repair (exr-), as compared to the wild-type (exr+); (ii) relative clastogenic efficiency, expressed by the ratio of chromosomal aberrations (ring-X loss) to SLRL determined in the exr+ strain. For a subset of direct-acting, monofunctional alkylating agents, nucleophilic selectivity and TD50 values or hypermutability indices were linearly correlated. In addition, the hypermutability indices in Drosophila by methylating or ethylating procarcinogens were similar to the corresponding values of their ultimate metabolites. In contrast, cross-linking agents, including antitumour drugs, did not show these positive correlations. The relative clastogenic efficiencies in Drosophila of 26 direct-acting, alkylating carcinogens increased with both their cross-linking activity and nucleophilic selectivity. By analyzing mutational spectra in Drosophila induced in the vermilion gene by four monofunctional alkylating agents with contrasting s values, critical DNA lesions, i.e. type of base pair substitution mutations, deletions, insertions, involved in genotoxicity were pinpointed. Thus, these multi-endpoint analyses should, as a new approach, assist in the quantitative risk evaluation of genotoxic agents.     

Control of carcinogenic hazards in industry.

Complete control of carcinogenic hazards in industry is an unrealistic concept. Industry, nevertheless, has a duty to give careful consideration to existing carcinogenic hazards, and to the possibility of new ones. Only well recognized hazards can be subjected to legislative control. Industry must take into account published experimental and epidemiological data, along with physical and chemical considerations related to specific agents, and, where necessary, introduce appropriate preventive measures.     

Carcinogenesis and its modification by environmental endocrine disruptors: in vivo experimental and epidemiological findings

Although a great deal of concern has been raised about the hazard potential of endocrine disruptors present in the environment, the in vivo data available from both experimental and epidemiological studies suggest that the majority of those agents do not pose a risk with regard to cancer development. Indeed, naturally occurring examples such as isoflavonoids even appear to exert protective effects. Only for xenobiotics such as 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), polychlorinated biphenyls (PCBs) and tetrachloro-p-dioxin (TCDD) and special cases of phenols and phthalates is there unequivocal evidence of carcinogenicity and this appears to be directly linked to their toxicity. Thus, careful in vivo assessment is required before drawing any conclusions regarding agents capable of affecting the mammalian endocrine system.     

Natural pesticides present in edible plants are predicted to be carcinogenic

Based upon the US National Toxicology Program (NTP) rodent carcinogenicity data base, CASE, an artificial intelligence structure-activity evaluation method, predicts that a large proportion of natural pesticides present in edible plants are rodent carcinogens.     

Many roads lead to oncogene-induced senescence

Oncogene-induced senescence is a mechanism of tumor suppression that restricts the progression of benign tumors. Important advances have been made toward elucidating the mechanisms that regulate this response; however, there is presently no unified model that integrates all current findings. DNA damage, replicative stress, reactive oxygen species, heterochromatin formation and negative feedback signaling networks have all been proposed to play an integral role in promoting senescence in response to various oncogenic insults. In all cases, these signals have been shown to function through Rb and p53, but utilize different intermediaries. Thus, it appears that senescence is not triggered by a single, linear series of events, but instead is regulated by a complex signaling network. Accordingly, multiple proteins may cooperate to establish a senescence response, but the limiting signal(s) may be dictated by the initiating genetic alteration and/or tissue type. This review will focus on integrating current models and will highlight data that provide new insight into the signals that function to suppress human tumor development.     

The carcinogenic role of oncogenic HPV and p53 gene mutation in cervical adenocarcinomas

Thirty tumors were collected from our archive of cervical adenocarcinomas. They were examined with respect to the content of oncogenic HPV and presence of mutations in the p53 gene exons 5 through 8. Furthermore, available clinical information on the cases was reviewed. For the detection of p53 gene and presence of oncogenic HPV, PCR followed by direct sequence analysis of the amplified DNA was employed. Seventeen tumors were identified as HPV-positive, comprising both HPV types 18 and 16. Six cases showed a p53 gene mutation, of which five were of the missence and one of the silent type. No statistical correlation between the occurrence of oncogenic HPV and presence of p53 gene mutation (p=0.67) was recorded. Among the tumors with p53 gene mutation, three were HPV-positive and three were HPV-negative. The determination of p53 gene mutations was not related to clinical findings such as the stage of the tumor or presence of metastases of the lymph nodes. However, p53 gene mutations were somewhat more prevalent in low differentiated tumors (p<0.02). The results indicate that oncogenic HPV and p53 gene mutations have independent carcinogenic roles in cervical adenocarcinomas.