Dose-response and threshold effects in cytotoxicity and apoptosis

Cell death can occur as an active, programmed event in response to cytotoxic injury or to endogenous growth limiting factors; the latter serve to maintain homeostasis of cell number in tissues. Cells seem to use different pathways for programmed death, as reflected by their different morphology and different biochemistry. Severe cell damage leading to incapacitation of essential cell functions such as ATP synthesis or the maintenance of membrane potential may lead to "necrosis". In any event, the incidence and rate of cell death increase with increasing signal intensity. Cytotoxic injury requires a certain number of primary insults; cell death will therefore occur only beyond a definable threshold. Growth factor control of cell death is receptor-mediated with dose-response relations including threshold phenomena follow the general principles of receptor kinetics. The occurrence of programmed cell death during the stages of carcinogenesis introduces a reversible component into this disease. Therefore, there may exist thresholds of dose or durations of exposure to certain carcinogens below which irreversible disease is not generated.     

Dose-response and threshold-mediated mechanisms in mutagenesis: statistical models and study design

The objective of this paper is to review the use, in mutagenesis, of various mathematical models to describe the dose-response relationship and to try to identify thresholds. It is often taken as axiomatic that genotoxic carcinogens could damage DNA at any level of exposure, leading to a mutation, and that this could ultimately result in tumour development. This has led to the assumption that for genotoxic chemicals, there is no discernible threshold. This assumption is increasingly being challenged in the case of aneugens. The distinction between 'absolute' and 'pragmatic' thresholds is made and the difficulties in determining 'absolute' thresholds using hypothesis testing approaches are described. The potential of approaches, based upon estimation rather than statistical significance for the characterization of dose-response relationships, is stressed. The achievement of a good fit of a mathematical model to experimental data is not proof that the mechanism supposedly underlying this model is operating. It has been argued, in the case of genotoxic chemicals, that any effects produced by a genotoxic chemical which augments that producing a background incidence in unexposed individuals will lead to a dose-response relationship that is non-thresholded and is linear at low doses. The assumptions underlying this presumption are explored in the context of the increasing knowledge of the mechanistic basis of mutagenicity and carcinogenicity. The possibility that exposure to low levels of genotoxic chemicals may induce and enhance defence and repair mechanisms is not easily incorporated into many of the existing mathematical models and should be an objective in the development of the next generation of biologically based dose-response (BB-DR) models. Studies aimed at detecting or characterizing non-linearities in the dose-response relationship need appropriate experimental designs with careful attention to the choice of biomarker, number and selection of dose levels, optimum allocation of experimental units and appropriate levels of replication within and repetition of experiments. The characterization of dose-response relationships with appropriate measures of uncertainty can help to identify 'pragmatic' thresholds based upon biologically relevant criteria which can help in the regulatory process.     

Specific-locus mutations induced in eukaryotes (especially mammalian cells) by radiation and chemicals: a perspective

In the course of discovering the first mutagen (X-rays) just over 60 years ago, Herman J. Muller asked whether X-rays induced single-gene mutations and/or chromosomal (multiple-gene) mutations. To a large extent, his question has set the agenda for mutagenesis research ever since. We explore historically the answers to this question, with special emphasis on recent developments in the field of mammalian cell mutagenesis. Studies indicate that ionizing radiation and many chemical mutagens/carcinogens induce both gene and chromosomal mutations; however, only certain genetic systems permit the recovery and analysis of both classes of mutations. Few chemical mutagens induce only gene mutations in mammalian cells; instead, most mutagens appear to induce both classes of mutations, with chromosomal mutations (especially multilocus deletions) predominating at high doses. These results have implications regarding the mechanisms of mutagenesis, the role of chromosomal mutations in carcinogenesis and hereditary disease, and the type of data required for risk assessment of physical and chemical mutagens/carcinogens.     

Molecular dosimetry studies of forward mutation induced at the yg2 locus in maize by ethyl methanesulfonate

The yg2 assay in Zea mays detects forward mutation in somatic cells within leaf primordia of embryos and it was used in an analysis of the molecular dosimetry of ethyl methanesulfonate (EMS). Parallel genetic and molecular dosimetry experiments were conducted in which the frequency of forward mutation and the level of covalently bound ethyl DNA adducts were determined. Prepared kernels were treated for 8 h at 20 degrees C with 1-10 mM EMS. EMS induced a direct concentration-dependent increase in mutation induction proportional to the exposure concentration (slope = 0.93). The kinetics of mutation induction demonstrated in the intact maize system were consistent with the kinetics observed earlier in in vitro model systems using cultured mammalian cells, and contrasted with the exponential increase in mutation induction characteristic of microbial species. Parallel molecular dosimetry experiments were conducted using [3H]EMS. DNA was extracted and purified from embryonic tissues containing the leaf primordia, the target tissue of the yg2 assay. A linear increase in the molecular dose was observed as a function of EMS concentration. Using concentration as a common parameter between the parallel genetic and dosimetry studies, mutation induction appeared to increase nearly in a direct proportion to the molecular dose. However, studies in other genetic systems indicate that the levels of specific DNA adducts, such as O6-ethylguanine (O6-EtGua) show a better correlation with mutation induction kinetics than molecular dose. Neither molecular dose, nor O6-EtGua levels account for differences in the absolute frequencies of mutation induction observed in different genetic systems. Therefore, reliable assessment of health risks posed to humans by chemical mutagens appears to require consideration of other factors in addition to DNA dose or adduct formation, including differences in repair capabilities and in the size of the genetic targets in humans relative to the model genetic systems under study.     

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.     

Model-Based Statistical Procedures for the Analysis of in Vitro Mutagenesis Assays

A number of short-term screening assays for potential chemical carcinogens and mutagens involve the measurement of mutation rates in in vitro cell populations. In this paper, statistical procedures are proposed for the analysis of results from these assays. The procedures, which are based on previously developed stochastic models of cell growth and mutation, include hypothesis tests for the comparison of mutation rates in treated and control cultures, and estimation and hypothesis tests for the parameters of a linear mutagenesis dose-response. Computer simulation is used to validate the methods, and they are illustrated by an analysis of data obtained under the mouse lymphoma mutagenesis protocol.     

Repair of exocyclic DNA adducts: rings of complexity

Exocyclic DNA adducts are mutagenic lesions that can be formed by both exogenous and endogenous mutagens/carcinogens. These adducts are structurally analogs but can differ in certain features such as ring size, conjugation, planarity and substitution. Although the information on the biological role of the repair activities for these adducts is largely unknown, considerable progress has been made on their reaction mechanisms, substrate specificities and kinetic properties that are affected by adduct structures. At least four different mechanisms appear to have evolved for the removal of specific exocyclic adducts. These include base excision repair, nucleotide excision repair, mismatch repair, and AP endonuclease-mediated repair. This overview highlights the recent progress in such areas with emphasis on structure-activity relationships. It is also apparent that more information is needed for a better understanding of the biological and structural implications of exocyclic adducts and their repair.     

Some chemical aspects of dose-response relationships in alkylation mutagenesis

Alkylation of DNA can lead to induction of potentially miscoding groups (promutagenic) or potentially template-inactivating groups (lethal). The proportions of these are found to vary with the chemical nature of the alkylating agent. Agents of low Swain and Scott s factor (or those tending to Ingold's S_Ni type) react relatively more extensively at O-atom sites in DNA, and yield relatively more of the miscoding O₆-alkylguanine residues. Phosphotriester formation is also relatively more extensive with S_Ni agents.Inactivation of DNA can result from depurinations, strand breakage, and cross-linkage.Both promutagenic and lethal lesions are subject to repair; 3 principal enzymatic systems appear to exist; one for excision and repair of cross-links or aralkyl groups resembles the uvr system; others for repair of single-strand breaks parallel repair of X-ray-induced breaks (exr, rec systems); another, less well defined at present, recognizes certain methylated bases, and depurinated sites (probably Goldthwait's endonuclease II).These factors can be shown to influence dose-response in alkylation mutagenesis. This, broadly, can be classified as linear with the promutagenic group-inducing or directly miscoding agents, and is independent of cytotoxicity; whereas with other agents non-linear response parallels the occurrence of “shouldered” survival curves, and reflects mutation induction by “repairs errors”.Additionally, alkylation of cellular constituents other than DNA, e.g. repair enzymes, may influence dose response, and will again depend on chemical reactivity of the agent.     

Carcinogen-induced inflammation and immunosuppression are enhanced in xeroderma pigmentosum group A model mice associated with hyperproduction of prostaglandin E2

Xeroderma pigmentosum group A (XPA) gene-deficient mice easily developed skin cancers by the application of topical chemical carcinogens as well as by UV irradiation. As certain chemical carcinogens have been shown to be immunosuppressive, we examined the inflammatory and immunosuppressive effects of dimethylbenz(a)anthracene (DMBA) on XPA mice. Compared with wild-type mice, XPA mice showed greater ear swelling and reduction of epidermal Langerhans cells after DMBA application. Topical application of DMBA impaired the induction of contact hypersensitivity, initiated either locally or at distant sites. These DMBA-induced local and systemic immunosuppressions were more greatly enhanced in XPA mice than in wild-type mice. DMBA application induced pronounced production of PGE(2), IL-10, and TNF-alpha in the skin of XPA mice. Treatment with indomethacin, a potent inhibitor of PG biosynthesis, inhibited DMBA-induced inflammation and local immunosuppression. In XPA mice, increased serum IL-10 was detected after DMBA treatment. Excess production of PGE(2), TNF-alpha, and IL-10 after DMBA application may be involved in the enhanced local and systemic immunosuppression in DMBA-treated XPA mice. Susceptibility to DMBA-induced skin tumors in XPA mice may be due to easy impairment of the immune system by DMBA in addition to a defect in the repair of DMBA-DNA adduct. Enhanced immunosuppression by chemical carcinogens as well as the mutagenicity of these mutagens might be associated with the high incidence of internal malignancies seen in XP patients. Moreover, these results supported the hypothesis that persistent DNA damage is a trigger for the production of immunoregulatory cytokines.     

Chemical induction of streptomycin-resistant mutations in Escherichia coli. Dose and mutagenic effects of dichlorvos and methyl methanesulfonate

A procedure for the quantitative determination of induced streptomycin-resistant mutants in E. coli was applied to study and compare mutation induction by the organophosphate dichlorvos and by methyl methanesulfonate (MMS). Both compounds increased the frequency of mutants even under conditions where no inactivation of cell was observed. Mutation induction by these agents as a function of both concentration and exposure time was measured. The dose-response curves found with both mutagens were non-linear; atp higher doses more mutants were induced per unit dose than at lower doses. Possible relationships between dose-effect curves and the chemical nature of alkylating mutagenic agents are discussed.     

Quantitative evaluation of the effectiveness of the formation of sister chromatid exchanges and chromosome aberrations as affected by chemical mutagens

Dose curves of five chemicals were studied to compare the efficiency of SCE and chromosomal aberration induction by different chemical mutagens. SCEs were found to increase linearly with the dose, whereas chromosomal aberrations--nonlinearly. Using regression coefficients obtained from the dose curves it was found that the efficiency of the studied chemical mutagens in induction of SCEs is 100-300 times as high as that in the induction of chromosomal aberrations.     

The induction of thymidine- and IUdR-resistant variants in P388 mouse lymphoma cells by x-rays, UV and mono- and bi-functional alkylating agents

Dose-response curves for “mutation” to resistance to 5-iodo-2-deoxyuridine (IUdR) and excess thymidine (TdR) in P388 mouse lymphoma cells have been established after exposure of these cells to six chemical mutagens, UV |and| ionising radiations. The dose-response curves for all mutagens in both selective system show considerable similarities when induced mutation frequencies are plotted against survival. Expression time for both types of variants, IUdR^r and TdR^r, are similar, i.e. maximum frequencies are reached by 48 h and there is no fall in variant frequency at late expression times up to 144 h. Over the range of survival levels studied there appears to be little or no dependence of expression time on dose of mutagen. Some loss of mutants after high doses (i.e. at low survival levels) was observed due to the fact that a significant proportion of both TdR^r and IUdR^r clones were more sensitive to the mutagens than the wild-type population. The similarities in induced dose-response curves for different mutagens suggest that the mutants have a common origin, probably an error in repair, but it seems unlikely that errors in “cut and patch” repair are responsible. A comparison of spontaneous frequencies of IUdR^r and TdR^r variants suggests that IUdR is mutagenic in P388 cells.     

Comparison of in vivo and in vitro SCE induction

In vivo and in vitrod sister-chromatid exchange (SCE) induction and cell replication kinetics were compared in P388 cells exposed to 4 mutagens. While concordance was observed between SCE induction and inhibition of cell replication kinetics, certain mutagens were more potent in vivo while others were more potent in vitro. These results indicate that caution should be applied before equating in vivo and in vitro mutagen exposures.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.     

Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicity of methylating agents

Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells. Their effects can be ascribed to an ability to modify DNA covalently. Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O(6)-methylguanine (O(6)meG) as a potentially important DNA lesion. Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells - including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli - have defined the contributions of O(6)meG and other methylated DNA bases to the biological effects of these chemicals. More recently, the role of O(6)meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O(6)meG and the mismatch repair pathway. Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O(6)meG. We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent. Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive. Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens.     

In vivo sister-chromatid exchange: A sensitive measure of DNA damage

A variety of chemical agents and X-irradiation were examined for their abilities to induced sister-chromatid exchanges (SCE) in vivo. In addition to demonstrating the several known mutagens and carcinogens are capable of inducing SCE in vivo, our studies indicate that the suspected carcinogen, tris-bromophosphate, can significantly elevate SCE levels. Comparison of the effects of these agents on SCE levels, chromosomal-aberration frequencies and cell-replication kinetics reveals that no consistent relationship exists between SCE levels and other indicators of cellular DNA damage.

It is proposed that analysis of SCE induction in vivo may provide a useful technique for the screening of mutagenic and carcinogenic compounds.     

Susceptibility differences in chemical carcinogenesis linearize the dose-response relationship: threshold doses can be defined only for individuals

The debate on thresholds in dose-response relationships for chemical carcinogenesis concentrates on the question of mechanisms of action that come into play only at dose levels that overwhelm compensatory control mechanism, such as DNA repair or regulation of cell proliferation and death. In this article, individual susceptibility differences are introduced. It is postulated that one single threshold dose cannot be defined for a heterogeneous population because both the background rate of carcinogenesis and specific exposure-related effects differ between individuals. A threshold dose can therefore be defined only on an individual basis and for a given organ. Expressed as a time-to-tumor, the threshold dose results in tumor manifestation at exactly the end of the specified observation period. For those individuals who do not have cancer by the end of this period, the dose was below their individual threshold dose, for those who do have cancer, the dose was above their threshold dose. Based on this concept, a distinction between genotoxic and non-genotoxic carcinogens is no longer required; both types modulate time-to-tumor. Although the present analysis does not allow to define a threshold dose for a population, the setting of a "limit value" for regulatory purposes can be considered if regulators are aware of the fact that this splits a population at some percentile into a group for which the chosen standard is protective and a group for which it might not be. Investigation of factors that confer particular susceptibility to individuals is the key to an understanding of the dose-response relationship at low dose.     

DNA double-strand breaks in human lymphocytes after single irradiation by low doses of pulsed X-rays: non-linear dose-response relationship

Effects of ionizing radiation registered in cells after low dose irradiation are still poorly understood. A pulsed mode of irradiation is even more problematic in terms of predicting the radiation-induced response in cells. Thus, the aim of this paper was to study and analyze the effects of dose and frequency of pulsed X-rays on the frequency of radiation-induced DNA double-strand breaks and their repair kinetics in human peripheral blood lymphocytes in vitro. Analysis of radiation-induced gammaH2AX and 53BP1 repair foci was used to assess the DNA damage in these cells. The dose-response curve of radiation-induced foci of both proteins has shown deviations from linearity to a higher effect in the 12-32 mGy dose range and a lower effect at 72 mGy. The dose-response curve was linear at doses higher than 100 mGy. The number of radiation-induced gammaH2AX and 53BP1 foci depended on the frequency of X-ray pulses: the highest effect was registered at 13 pulses per second. Moreover, slower repair kinetics was observed for those foci induced by very low doses with a nonlinear dose-response relationship.