This paper describes the four cytogenetic endpoints most frequently used in hazard identification assays as the first step in the risk assessment process. These are structural chromosome aberrations, micronuclei, aneuploidy, and sister chromatid exchanges. The biological mechanisms involved in the formation of the alterations observed in each assay are briefly discussed. Variations in and recent improvements to each assay are described, with an emphasis on the use of molecular techniques to improve the sensitivity of the assay, and to allow for detection of specific alterations that are, or could be, associated with cancer induction. This, in turn, will make the data obtained in the cytogenetic assays more useful in cancer and genetic risk assessment. Thus, the aim of this paper is to encourage cytogeneticists to design their experiments in such a way that the data obtained will be of maximum possible benefit for characterizing and quantifying adverse human health effects, particularly cancer.
The response to ionising radiation, in terms of level of cell killing, depends on a number of factors that may be grouped into those that are genetically controlled, radiation quality and dosage, and environmental factors. There is a range of genetically controlled cellular properties such as stage of differentiation, mutations in specific genes (such as p53 and bcl-2) and stage of transformation that will determine the ability of the target cell to enter apoptosis. The so-called normal cells, are usually more radiosensitive and the majority of the cell population will enter into an apoptotic death. However, in response to high doses of ionising radiation and complex DNA damage as produced by high-LET radiation, an increased fraction of these cells will die by necrosis. There are several examples of environmental factors with relevance for the combined action of radiation and xenobiotics on carcinogenesis and in tumour therapy. In the case of normal cells, agents such as growth factors and tumour promoters, may decrease radiosensitivity. For certain type of tumour cells, radiation sensitivity can be increased in the presence of agents such as hormones, and the cells may die an apoptotic death. Removal of heavily compromised cells is essential to prevent a potential spreading of mutated clones. However, if apoptosis is inhibited (e.g., by tumour promoter), an increased fraction of damaged cells carrying genotoxic lesions may survive. This would significantly increase the risk of proliferation of precancerous cells. As discussed above, it is probably incorrect to make predictions about relative radiosensitivity based solely on mode of death. Intrinsic characteristics deriving from the cell type of origin of a line may be more important in determining radiosensitivity. The rapidly increasing knowledge about the process of radiation induced apoptosis has opened new frontiers in radiation biology, genetic toxicology, and cancer therapy and strongly motivates further research in this field.
The ability to quantify somatic mutations in vivo provides a new source of toxicological information that is relevant to the assessment of cancer risk. The major experimental factors that influence the mutant frequency are age, time after treatment, treatment protocol, and tissue analyzed. In untreated mice, the mutant frequency increases very rapidly with age from conception to birth, more slowly from birth to adulthood, and very slowly thereafter. All somatic tissues studied so far in adults have similar mutant frequencies. The time after treatment (expression time) is the most important experimental variable. The minimum time for expression varies from one tissue to another. To be valid, comparisons between tissues and treatments must be made after complete expression of the mutations. Unfortunately, the minimum expression time has not been characterized in most tissues. Since carcinogens are tissue specific, and many chemicals are distributed in the body in complex patterns, it is to be expected that there will be differences in the frequency of mutation induced in different tissues. As yet this has not been extensively studied. Since the mutations detected by the transgenic assays are neutral, the mutants should accumulate as the integral of the mutation rate. Hence chronic treatment protocols should be more effective than acute and subacute protocols whenever they permit substantially larger doses to be delivered. Such protocols are more relevant to human exposure and are preferable for dose extrapolations. The importance of transcription in determining mutation rates is not yet known, but it is noteworthy that the transgenes are not transcribed whereas the Ioci involved in carcinogenesis are. The mutation spectrum is important for quantitative risk estimation. Risk estimation must also take into account the spectrum of mutations that are involved in the carcinogenic process in the tissue and the spectrum of mutations that are detectable by the assay. New assays are being used to quantify mutations in vivo in order to understand the carcinogenic process, to search for the environmental factors involved in human cancer, and to evaluate the carcinogenic hazard qualitatively.
Spatial factors conditioning the formation of radiation-induced chromosome exchange aberrations are reviewed, and concepts such as 'rejoining distance' and 'site' are re-examined in the light of the unexpectedly high frequencies of multi-break ('Complex') exchanges being revealed by FISH painting. Given the anticipated densities of dsb within a nucleus, and assuming random 3-D break distribution, nearest-neighbour analysis indicates that the most likely break interaction distance is a well defined shell, several hundred nm from each break. The sharpness with which this shell is defined increases with break density, and therefore with dose. It is argued that random movement and chance meeting over such distances will not account for the Complex frequencies observed, and that other factors, or modes of formation, must be invoked.
Apoptosis is one form of physiological or active cell death. The balance between cell proliferation and cell death or apoptosis not only effects organ growth but also has a profound impact on the net increase and growth of initiated cells and preneoplastic and tumor cell populations. With respect to cancer development apoptosis is becoming widely recognized as being an innate tissue defense against carcinogens by inhibiting survival and controlling growth of precancerous cell populations and tumors at different stages of carcinogenesis. Experimental data on cell birth and cell death rates help identify the mode of action of a chemical and can be incorporated into biologically based cancer models. This article describes the quantitation and regulation of apoptosis in rodent liver and how loss of regulation can have a role in hepatocarcinogenesis. A biologically-based mouse liver cancer model is presented and utilized to describe how treatment related growth effects affect the process of carcinogenesis. Advantages and limitations of biologically based cancer models in cancer research and risk assessment are discussed.
Data supporting the use of transgenic lines to identify carcinogens and noncarcinogens are thus far based on a limited number of chemicals for which there are also long-term bioassay results in rats and/or mice. Six chemicals have been tested in the heterozygous p53-deficient mice and 13 in the Tg . AC line. The results show that the p53(def) responds rapidly to mutagenic carcinogens and the Tg . AC responds rapidly to both mutagenic and nonmutagenic carcinogens. Neither transgenic line responded to the noncarcinogens that were tested. The p53(def) line failed to respond to two nonmutagenic carcinogens (N-methyloacrylamide and reserpine), the Tg . AC line failed to respond to ethyl acrylate, a nonmutagenic chemical that induced tumors of the forestomach when administered by gavage, and to triethanolamine that caused an increase in hepatocellular tumors in B6C3F1 mice via skin painting. Both of the latter chemicals are examples of highly specific responses related to either route of administration or to strain susceptibility. Further efforts to evaluate the range of chemicals to which these transgenic lines respond are currently in progress.
Three functional elements are required for the stable transmission of eukaryotic chromosomes: replication origins, centromeres and telomeres. In the yeast Saccharomyces cerivisiae the DNA sequences defining each of these elements are known. The simplest and most widely conserved of these sequences is that of the telomere. As the name implies, the telomere is the end of a linear eukaryotic chromosome. Two of the main functions of the telomere are to prevent DNA loss as a consequence of replication and to prevent interactions with other chromosomal ends. Thus, telomeres play a major role in maintaining chromosome stability and consequently they have been considered as likely to be involved in some aspects of chromosomal aberration formation. The involvement of telomeric DNA sequences in stabilizing normal and broken chromosome ends, in ‘hot spots’ for aberration formation and in delayed chromosomal instability will be reviewed here drawing on material presented at the Workshop and the published literature.
Analyses of a data base consisting of 461 chemicals tested in the SOS chromotest with MULTICASE resulted in the development of an SAR model that displayed a highly significant concordance (87.3%) between experimental and predicted results of chemicals not included in the model. An analysis of the nature of the biophores and their modulators revealed that electrophilicity and structural features affecting: (a) accessibility of the electrophile to the nucleophilic site on the DNA; and (b) the bulkiness of the DNA adduct were factors determining the probability that a chemical would induce DNA error prone repair and if so the extent of this activity. Additional analyses indicated that there were significant mechanistic similarities between the SOS chromotest and mutations in Salmonella as determined in the standard ('Ames') assay.
We compared the mechanism of action of micronuclei (MN), unstable chromosome aberrations, and 8-hydroxyde oxyguanosine (8-OHdG) levels to evaluate the genotoxicity of methyl mercuric chloride (CH3HgCl) and mercuric chloride (HgCl2) in human peripheral lymphocytes. The chromosome aberrations in human peripheral lymphocytes exposed to various concentrations of CH3HgCl or HgCl2 increased in a concentration-dependent manner and were significantly higher than the control when the cells were incubated with 1 X 10(-5) M (HgCl2) or 2 X 10(-6) M (CH3HgCl). The increase in the incidence of micronucleated lymphocytes was significant among the exposed groups, being 2 X 10(-5) M (HgCl2) and 5 X 10(-6) M (CH3HgCl) compared with the control. CH3HgCl was about 4-fold more potent than HgCl2. We determined the 8-OHdG levels in human peripheral blood mononuclear cells(PBMC) and found that they were significantly higher in the exposed groups at 1 X 10(-5) M (HgCl2) and 5 X 10(-6) M (CH3HgCl) compared with the control. A detectable (p < 0.05) increase in the level of 8-OHdG was induced by CH3HgCl at a concentration that was about 50% of the amount of HgCl2 required to produce a similar response. The data confirmed the value of the MN and/or chromosome aberration assays for assessing of HgCl2- and/or CH3HgCl-induced genotoxicity, and indicated that they are about the same concentration as the 8-OHdG assay. The presence of genotoxic effects in peripheral blood lymphocytes exposed to the mercuric compounds indicated by the chromosome aberrations and the MN assays could be partly due either to the disturbance of the spindle mechanism, or to the elevated level of 8-OHdG brought by the generation of reactive oxygen species.
An extremely large database describes genotypes associated with the human cancer phenotype and genotypes of human populations with genetic predisposition to cancer. Aspects of this database are examined from the perspective of risk analysis, and the following conclusions and hypotheses are proposed: (1) The genotypes of human cancer cells are characterized by multiple mutated genes. Each type of cancer is characterized by a set of mutated genes, a subset from a total of more than 80 genes, that varies between tissue types and between different tumors from the same tissue. No single cancer-associated gene nor carcinogenic pathway appears suitable as an overall indicator whose induction serves as a quantitative marker for risk analysis. (2) Genetic defects that predispose human populations to cancer are numerous and diverse, and provide a model for associating cancer rates with induced genetic changes. As these syndromes contribute significantly to the overall cancer rate, risk analysis should include an estimation of the effect of putative carcinogens on individuals with genetic predisposition. (3) Gene activation and inactivation events are observed in the cancer genotype at different frequencies, and the potency of carcinogens to induce these events varies significantly. There is a paradox between the observed frequency for induction of single mutational events in test systems and the frequency of multiple events in a single cancer cell, suggesting events are not independent. Quantitative prediction of cancer risk will depend on identifying rate-limiting events in carcinogenesis. Hyperproliferation and hypermutation may be such events. (4) Four sets of data suggest that hypermutation may be an important carcinogenic process. Current mechanisms of risk analysis do not properly evaluate the potency of putative carcinogens to induce the hypermutable state or to increase mutation in hypermutable cells. (5) High-dose exposure to carcinogens in model systems changes patterns of gene expression and may induce protective effects through delay in cell progression and other processes that affect mutagenesis and toxicity. Paradigms in risk analysis that require extrapolation over wide ranges of exposure levels may be flawed mechanistically and may underestimate carcinogenic effects of test agents at environmental levels. Characteristics of the human cancer genotype suggest that approaches to risk analysis must be broadened to consider the multiplicity of carcinogenic pathways and the relative roles of hyperproliferation and hypermutation. Further, estimation of risk to general human populations must consider effects on hypersusceptible individuals. The extrapolation of effects over wide exposure levels is an imprecise process.
Chronic kidney disease (CKD) is one of the most prevalent chronic diseases and affects between 10 and 14 % of the world's population. The World Health Organization estimates that by 2040, the disease will be fifth in prevalence. End-stage CKD is characterized by renal fibrosis, which can eventually lead to kidney failure and death. Renal fibrosis develops due to multiple injuries and involves oxidative stress and inflammation. In the human body, nuclear factor erythroid 2-related factor 2 (Nrf2) plays an important role in the expression of antioxidant, anti-inflammatory, and cytoprotective genes, which prevents oxidative stress and inflammation damage. Heme oxygenase (HO-1) is an inducible homolog influenced by heme products and after exposure to cellular stress inducers such as oxidants, inflammatory chemokines/cytokines, and tissue damage as an outcome or downstream of Nrf2 activation. HO-1 is known for its antioxidative properties, which play an important role in regulating oxidative stress. In renal diseases-induced tissue fibrosis and xenobiotics-induced renal fibrosis, Nrf2/HO-1 has been targeted with promising results. This review summarizes these studies and highlights the interesting bioactive compounds that may assist in attenuating renal fibrosis mediated by HO-1 activation. In conclusion, Nrf2/HO-1 signal activation could have a renoprotective effect strategy against CKD caused by oxidative stress, inflammation, and consequent renal fibrosis.
Results from short-term tests for carcinogens and our advanced knowledge on cellular and molecular mechanisms of carcinogenesis strongly suggest that carcinogens do not induce genetic changes necessarily by directly interacting with DNA. Therefore, it is not surprising to see that many carcinogens are not detectable by available genetic toxicology tests. Thus, it has become necessary to study nongenotoxic mechanisms of carcinogenesis and to provide methods to predict those carcinogens which escape from conventional mutation tests. One possible nongenotoxic mechanism of carcinogenesis which is supported by abundant experimental evidence is inhibition of gap junctional intercellular communication. Many, but not all, tumor-promoting agents have been shown to inhibit the communication of cultured cells as well as in vivo. Molecular mechanisms of gap junctional intercellular communication control revealed that connexin (gap junction) genes form a family of tumor suppressor genes. Control mechanisms of expression as well as function of connexins are vulnerable to various carcinogenic insults, notably to nongenetoxic carcinogens. Thus, studies on the role of connexins in cell growth and carcinogenesis may prove to be useful for establishing a mechanism-based test to detect certain types of nongenotoxic carcinogens.
The epidemiology of multiple sclerosis (MS) exhibits significant variability all over the world. Additionally, the incidence and prevalence of the disease are geographically diverse in the different provinces of Iran.Due to the lack of a research about the epidemiology of MS in the Markazi province of Iran, the present study aimed to estimate its prevalence and incidence in this province.This cross-sectional register study was conducted according to the data related to the MS Society of Markazi province. The inclusion criteria were definite MS during 2010–2019, residence in Markazi province, and membership in the MS society of Markazi province. The annual incidence and prevalence of MS (per 100,000 person-years) were computed by sex and age groups. Further, age-standardized prevalence and incidence rates were calculated based on the Iranian population. A gamma regression model with log link was utilized for comparing the rates over time statistically. The prevalence and incidence rates were computed by using Excel, and R 4.0.5 was applied to fit the gamma model.In the study, the total number of MS cases was 1,391, among whom 1,098 (78.9%) and 293 (21.1%) were females and males, respectively. The onset of MS occurred at the mean (S.D) age of 31.76 (8.98) years with a female/male ratio of 3.75. During the study period (2010–2019), the incidence rate of the disease was on the decline, while the prevalence rate elevated, by indicating a rising trend in MS prevalence in both sexes.The results represented the decreasing and increasing trend of the incidence and prevalence of MS in Markazi province over the recent years. The province is one of the regions with a high prevalence and incidence rate of MS compared to the national and global statistical data.
DNA adducts have been investigated extensively during the past decade. This research has been advanced, in part, by the development of ultrasensitive analytical methods, such as 32P-postlabeling and mass spectrometry, that enable detection of DNA adducts at concentrations as low as one adduct per 109 to 1010 normal nucleotides. Studies of mutations in activated oncogenes such as ras, inactivated tumor suppressor genes such as p53, and surrogate genes such as hprt provide linkage between DNA adducts and carcinogenesis. The measurement of DNA adducts, or molecular dosimetry, has important applications for cancer risk assessment. Cancer risk assessment currently involves estimating the probable effects of carcinogens in humans based on results of animal bioassays. Estimates of risk are then derived from mathematical models that fit data of tumor incidence at the high animal exposures and extrapolate to probable human exposures that may be orders of magnitude lower. Molecular dosimetry could extend the observable range of mechanistic data several orders of magnitude lower than can be achieved in carcinogenesis bioassays. This measurement also compensates automatically for individual and species differences in toxicokinetic factors, as well as any nonlinearities that affect the quantitative relationships between exposure and molecular dose. As a result, molecular dosimetry can provide a basis for conducting high- to low-dose, route-to-route, and interspecies extrapolations. The incorporation of such data into risk assessment promises to reduce uncertainties and produce more accurate estimates of risk compared to current methods.
Aflatoxin B-1 (AFB(1)) is classified as a group I carcinogen in humans by IARC. However, the exact mechanisms of AFB(1) hepatocarcinogenesis have not been fully elucidated. Recent studies have suggested that oncogenes are critical molecular targets for AFB(1), and AFB(1) causes characteristic genetic changes in the p53 tumor suppressor gene and ras protooncogenes. Up to date, more than 1500 human hepatocellular carcinoma (HCC) samples have been examined for p53 mutations with respect to different AFB(1) exposure levels. The most significant finding is that more than 50% of HCC patients from high aflatoxin exposure areas such as southern Africa and Qidong, China harboured a codon 249 G to T transversion in the p53 tumor suppressor gene, which is found to be consistent with the mutagenic specificity of AFB(1) observed in vitro. In contrast, this mutational pattern is not found in HCC samples from moderate or low aflatoxin exposure countries or regions. Therefore, this hot-spot mutation is believed to be a molecular fingerprint linking the initial event of AFB(1)-DNA adduct formation with the ultimate development and progress of human HCC. However, some important points still remain to be explicated. First, in many of these studies, the systematic evaluation of AFB(1) exposure is rather limited and the classification of AFB(1) exposure level is speculative and confusing, without the definite evidence for the actual aflatoxin exposure level. Second, the role of hepadnaviral infection has to be considered in the induction of this unique mutational spectrum. On the other hand, ras oncogene mutations are frequently found in AFB(1)-induced HCC samples in experimental animals, while the frequency of ras mutation in human HCC in contrast is much lower than that of p53, Recent studies have provided additional evidence that reactive oxygen species (ROS) and oxidative DNA damage may be involved in AFB(1)-induced p53 and ras mutations. In future, follow-up cohorts exposed to different levels of AFB(1) combined with the determination of putative gene markers are much needed.
Benzodiazepines are a group of drugs which have been extensively used for their activities as an anti-anxiety, sedative, muscle relaxant and anti-convulsant. Benzodiazepines at present are the most commonly prescribed drugs. Some of these drugs are teratogenic and also carcinogenic in experimental animals. The wide human exposure to this group of drugs throughout the world is of great concern for human health. In the present review, we have attempted to evaluate and update the mutagenic and genotoxic effects of four of the most commonly used benzodiazepines, i.e., chlordiazepoxide (CDZ), diazepam (DZ), nitrazepam (NZ) and oxazepam (OZ) based on available literature.
Recent years have seen the rapid expansion of scientific understanding of the underlying biologic bases of toxic reactions to chemicals. Use of this information in health risk assessment is expanding, but it has yet to reach its full potential. This article considers what has successfully been done, what approaches are now being developed, and what impediments and difficulties have been encountered in attempts to bring case-specific, mechanistic toxicological information to bear on risk estimation. In hazard identification, mechanistic information can help explain the bearing of various empirical experimental results for inferring human hazard, can increase the sensitivity of detection, and can be considered in attempts to replace 2-year animal bioassays with hazard identification methods that rest on identifying key biological properties underlying carcinogenicity rather than relying only on the experimental observation of tumors. In carcinogen potency estimation, mechanistic information can potentially extend relevant observation to lower dose levels, provide the basis for choosing among empirically based dose-response models, lead to potency estimates through relationships with quantitative measures of short-term test outcomes, and can be considered as a basis for providing direct observation of the biological parameters in biologically based dose-response modeling.
Sensitive methods for quantifying DNA adducts from (i) benzo[a]pyrene (BP), (ii) alkylation exposure, and (iii) etheno(ε)-DNA adduct-forming chemicals were developed and applied to humans and animal models. The aims were to identify hitherto unknown sources and mechanisms of exogenous and endogenous DNA damage, to examine the effect of drug polymorphism on BP adduct levels, and to develop QSAR between tumorigenic potency, heritable genetic damage and structural elements of alkylating carcinogens (Vogel and Nivard (1994) Mutation Res., 395, 13–32). (i) BP-DNA adducts: An HPLC/fluorimetry assay suitable for measuring (+)-anti-BP-diol-epoxide (BPDE) adducts in human tissues and white blood cells (WBC) was developed (Alexandrov et al. (1992) Cancer Res., 52, 6248–6253). In smokers, a positive correlation was found between pulmonary CYP1A1-related catalytic activity (AHH) and the level of lung BPDE-DNA adducts. In coke oven workers, an enhancing effect of smoking on BPDE-adduct levels in WBC was demonstrated (Rojas et al. (1995) Carcinogenesis, 16, 1373–1376). (ii) 3-Alkyladenines (3-alkAde): Akylating carcinogens form 3-alkAde adducts in DNA which depurinate to yield 3-alkAde in urine, for which a detection method was developed (Friesen et al. (1991) Chem. Res. Toxicol., 4, 102–106; Prevost et al. (1990) Carcinogenesis, 11, 1747–1751), using immunoaffinity purification and GC-MS analysis. The usefulness of 3-alkAde analysis for the determination of the whole-body dose of alkylating agents derived from exogenous and endogenous sources was demonstrated. (iii) Etheno-DNA adduct-forming agents: Etheno(ε)-DNA base adducts (εdA, εdC, εdG) are promutagenic DNA lesions that are formed by occupational (vinyl halides) and environmental (urethane) carcinogens. An ultrasensitive detection method was developed (Nair et al. (1995) Carcinogenesis, 16, 613–617),
Inhibitory activity of tetracycline against ofloxacin- and fleroxacin-induced bleaching of green and etiolated Euglena gracilis was examined. Tetracycline hydrochloride in concentrations of 83-2079 microM in the light partially inhibited the bleaching activity of 83 microM ofloxacin and of 162 microM fleroxacin. In the dark, the TC inhibition of the fluoroquinolones-induced bleaching activity was most obvious, the white colony counts were all decreased. The total inhibition of bleaching was observed in 43 microM ofloxacin and 81 microM fleroxacin both in light and darkness. Cell growth was not significantly influenced by ofloxacin, fleroxacin and tetracycline in the light or darkness. Cell growth was not significantly influenced by ofloxacin, fleroxacin and tetracycline in the light or darkness. Inhibition of ofloxacin-induced Euglena bleaching by tetracycline was more effective in etiolated cells. TC at 0-416 microM did not influence the growth of ofloxacin (2.15 microM)-induced Salmonella typhimurium revertants.
Genotoxicity test batteries have become a standard tool for identifying chemicals that may have potential carcinogenic risk to humans. It is now apparent, however, that the use of genotoxicity batteries for assessing carcinogenic potential has limitations including an overall low specificity and a limited ability to detect carcinogens acting via 'nongenotoxic' mechanisms. In vitro cell transformation models, because they measure a chemical's ability to induce preneoplastic or neoplastic endpoints regardless of mechanism, may fulfil the current need for an in vitro biologically relevant model with increased predictiveness for determining carcinogenic potential. This review will focus on data demonstrating the similarities of chemically induced cell transformation in vitro to carcinogenesis in vivo. Furthermore, a growing database demonstrating a high overall correlation between cell transformation results with those of the rodent bioassay will also be discussed. Finally, the inclusion of cell transformation approaches for assessing the carcinogenic potential of chemicals relative to currently used genotoxicity batteries will be presented.
