Oxidative stress related DNA adducts in the liver of female rats fed with sunflower-, rapeseed-, olive- or coconut oil supplemented diets.

Both animal and epidemiological studies support an effect of fatty acid composition in the diet on cancer development, in particular on colon cancer. We investigated the modulating effect of supplementation of the diet of female F344 rats with sunflower-, rapeseed-, olive-, or coconut oil on the formation of the promutagenic, exocyclic DNA adducts in the liver, an organ where major metabolism of fatty acids takes place. 1,N(6)-ethenodeoxyadenosine (etheno-dA), 3,N(4)-ethenodeoxycytidine (etheno-dC) and 1,N(2)-propandodeoxyguanosine from 4-hydroxy-2-nonenal (HNE-dGp) were determined as markers for DNA-damage derived from lipid peroxidation products and markers for oxidative stress. 8-Oxo-deoxyguanosine (8-Oxo-dG) was also measured as direct oxidative stress marker. The body weight of the rats was not influenced by the four diets containing the different vegetable oils during the 4-week feeding period. Highest adduct levels of etheno-dC (430 +/- 181 adducts/10(9) parent bases), HNE-dGp (617 +/- 96 adducts/10(9) parent bases) and 8-Oxo-dG (37,400 +/- 12,200 adducts/10(9) parent bases) were seen in rats on sunflower oil diet (highest linoleic acid content). Highest adducts levels of etheno-dA (133 +/- 113 adducts/10(9) parent bases) were found in coconut oil diet (lowest content of linoleic acid). Weakly positive correlations between linoleic acid content in the four diet groups were only observed for levels of HNE-dGp and 8-Oxo-dG. Neither the diet based on olive oil (which contains mainly oleic acid) nor the diet based on rapeseed oil (containing alpha-linolenic acid) exerted any significant protective effect against oxidative DNA damage. Our results indicate that a high linoleic acid diet may contribute to oxidative stress in the liver of female rats leading to a marginal increase in oxidative DNA-damage.

Nonlinearity and thresholds in dose-response relationships for carcinogenicity due to sampling variation, logarithmic dose scaling, or small differences in individual susceptibility.

Nonlinear and threshold-like shapes of dose-response curves are often observed in tests for carcinogenicity. Here, we present three examples where an apparent threshold is spurious and can be misleading for low dose extrapolation and human cancer risk assessment. Case #1: For experiments that are not replicated, such as rodent bioassays for carcinogenicity, random variation can lead to misinterpretation of the result. This situation was simulated by 20 random binomial samplings of 50 animals per group, assuming a true linear dose response from 5% to 25% tumor incidence at arbitrary dose levels 0, 0.5, 1, 2, and 4. Linearity was suggested only by 8 of the 20 simulations. Four simulations did not reveal the carcinogenicity at all. Three exhibited thresholds, two showed a nonmonotonic behavior with a decrease at low dose, followed by a significant increase at high dose ("hormesis"). Case #2: Logarithmic representation of the dose axis transforms a straight line into a sublinear (up-bent) curve, which can be misinterpreted to indicate a threshold. This is most pronounced if the dose scale includes a wide low dose range. Linear regression of net tumor incidences and intersection with the dose axis results in an apparent threshold, even with an underlying true linear dose-incidence relationship. Case #3: Nonlinear shapes of dose-cancer incidence curves are rarely seen with epidemiological data in humans. The discrepancy to data in rodents may in part be explained by a wider span of individual susceptibilities for tumor induction in humans due to more diverse genetic background and modulation by co-carcinogenic lifestyle factors. Linear extrapolation of a human cancer risk could therefore be appropriate even if animal bioassays show nonlinearity.

DNA adducts of styrene-7,8-oxide in target and non-target organs for tumor induction in rat and mouse after repeated inhalation exposure to styrene.

Styrene by inhalation had been shown to increase the lung tumor incidence in mice at 20 ppm and higher, but was not carcinogenic in rats at up to 1000 ppm. Styrene-7,8-oxide, the major metabolic intermediate, has weak electrophilic reactivity. Therefore, DNA adduct formation was expected at a low level and a 32P-postlabeling method for a determination of the two regioisomeric 2'-deoxyguanosyl-O6-adducts at the alpha(7)- and beta(8)-positions had been established. The first question was whether DNA adducts could be measured in the rat at the end of the 2 years exposure of a bioassay for carcinogenicity, even though tumor incidence was not increased. Liver samples of male and female CD rats were available for DNA adduct analysis. Adducts were above the limit of detection only in the highest dose group (1000 ppm), with median levels of 9 and 8 adducts per 10(7) nucleotides in males and females, respectively (sum of alpha- and beta-adducts). The result indicates that the rat liver tolerated a relatively high steady-state level of styrene-induced DNA adducts without detectable increase in tumor formation. The second question was whether different DNA adduct levels in the lung of rats and mice could account for the species difference in tumor incidence. Groups of female CD-1 mice were exposed for 2 weeks to 0, 40, and 160 ppm styrene (6h per day; 5 days per week), female CD rats were exposed to 0 and 500 ppm. In none of the lung DNA samples were adducts above a limit of detection of 1 adduct per 10(7) DNA nucleotides. The data indicate that species- and organ-specific tumor induction by styrene is not reflected by DNA adduct levels determined in tissue homogenate. The particular susceptibility of the mouse lung might have to be based on other reactive metabolites and DNA adducts, indirect DNA damage and/or cell-type specific toxicity and tumor promotion.

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.

Chemoimmunotherapy for melanoma with dacarbazine and 2,4-dinitrochlorobenzene: results from a murine tumour model.

An empirically established chemoimmunotherapy that combines the epifocal application of the contact sensitizer dinitrochlorobenzene (DNCB) to cutaneous metastases with the systemic administration of dacarbazine (DTIC) yields high response rates and results in prolonged survival. However, despite the fact that this therapy has been in clinical use for several years, the mode of action still remains elusive. In order to overcome this limitation we established a murine model system. B16 melanoma cells were implanted subcutaneously in syngeneic C57BL/6 mice and treatment was started 7 days after. In a first set of experiments mice received intraperitoneal injections of DTIC followed by epifocal applications of DNCB 24 h later. Treatment significantly decreased tumour growth. In contrast, no significant effect was induced by DTIC or DNCB alone. Using this regimen, with varying doses of either DTIC or DNCB, we demonstrated that the therapeutic effect is dose dependent. Furthermore, the treatment of subcutaneous tumours with DTIC and DNCB influenced the course of visceral metastases: the growth of pulmonary metastases was significantly inhibited if subcutaneous tumours were treated as described. In conclusion, we have established a model system that seems to be appropriate for both the optimization of this therapeutic regimen and the characterization of effector mechanisms.

Scientific analysis of the proposed uses of the T25 dose descriptor in chemical carcinogen regulation.

The uncertainties that surround the methods used for risk assessment of exposure to carcinogens have been highlighted by a recent document advocating an approach based on the T25 dose (the dose giving a 25% incidence of cancer in an appropriately designed animal experiment). This method relies on derivation of the T25 dose then assesses risk at the exposure dose using proportionality provided by a linear extrapolation (T25/linear). To promote discussion of the scientific issues underlying methods for the risk assessment of chemical carcinogens, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) hosted a one-day workshop in Brussels on 10 November 2000. Several invited presentations were made to participants, including scientists from regulatory authorities, industry and academia. In general, it was felt that there was sufficient basis for using the T25 dose as an index of carcinogenic potency and hence as part of the hazard assessment process. However, the use of the T25 in risk assessment has not been validated. The T25/linear and other extrapolation methods based on metrics such as LED 10 assume linearity which may be invalid. Any risk calculated using the T25/linear method would provide a precise risk figure similar to the output obtained from the Linearised Multistage (LMS) method formerly used by the Environmental Protection Agency (EPA) in the United States of America. Similarity of output does not provide validation but rather reflects their reliance on similar mathematical approaches. In addition to the T25 issue, evidence was provided that using two separate methods (linearised non-threshold model for genotoxic carcinogens; no-observable-effect level with a safety factor (NOEL/SF) method for all other toxicity including non-genotoxic carcinogens) is not justified. Since the ultimate purpose of risk assessment is to provide reliable information to risk managers and the public, there was strong support at the workshop for harmonisation of approaches to risk assessment for all genotoxic and nongenotoxic carcinogens. In summary, the T25 method has utility for ranking potency to focus efforts in risk reduction. However, uncertainties such as the false assumption of precision and non-linearity in the dose-response curve for tumour induction raise serious concerns that caution against the use of T25/linear method for predicting human cancer risk.

Supra-additive genotoxicity of a combination of gamma-irradiation and ethyl methanesulfonate in mouse lymphoma L5178Y cells.

While testing for genotoxicity is usually performed on single chemicals, exposure of humans always comprises a number of genotoxic agents. The investigation of potentially synergistic effects of combinations therefore is an important issue in toxicology. Combinations of 511 keV gamma-radiation with the chemical alkylating agent ethyl methane-sulfonate were investigated in the in vitro micronucleus test in mouse lymphoma L5178Y cells. With combinations in the low dose linear effect range for the individual agents (0. 25-2 Gy and 0.8-3.2 mM, respectively), supra-additivity by 34-86% was seen. The synergism was more pronounced at the higher dose levels. Supra-additivity was confirmed in experiments using cytochalasin B and analyzing binucleate cells only, to control for putative effects on the cell cycle. Statistical significance was shown by a 2-factor analysis of variance with interaction. The results indicate that damage to DNA by gamma-radiation and alkylation could affect different rate limiting steps in the formation of micronuclei. Further investigations will have to show whether the observations are of general validity, in particular, whether other end-points of genotoxicity produce the same results and whether the degree of supra-additivity is always dose dependent. The latter would have a strong impact on risk assessment for mixtures at low doses.

A true threshold dose in chemical carcinogenesis cannot be defined for a population, irrespective of the mode of action.

Strongly sigmoidal (S-shaped) dose-cancer incidence relationships are often observed in animal bioassays for carcinogenicity. If a genotoxic contribution is not plausible, an epigenetic mode of carcinogen action is proposed and a thresholded low dose-response suggested. In a strict sense, a threshold implies a no-yes situation, i.e., no effect up to the threshold dose and an effect above the threshold dose. A convincing explanation of the discontinuity of the gradient of the dose-response curve at the threshold dose is not available to me. However, the existence of a threshold is accepted for an individual. The threshold dose is the dose required for the manifestation of the tumor in an individual exactly at the end of a defined period of observation (for instance, 2 years in an animal bioassay, 75 years in humans). Because of genetic and lifestyle-dependent susceptibility differences, each animal or human has his individual threshold dose. For a group, no single threshold dose can be defined, irrespective of the mode of carcinogen action. Furthermore, in view of the stochastic elements in the process of carcinogenesis, the exact threshold dose can only be defined after tumor incidence and cannot be predicted.

Occurrence of emodin, chrysophanol and physcion in vegetables, herbs and liquors. Genotoxicity and anti-genotoxicity of the anthraquinones and of the whole plants.

1,8-Dihydroxyanthraquinones, present in laxatives, fungi imperfecti, Chinese herbs and possibly vegetables, are in debate as human carcinogens. We screened a variety of vegetables (cabbage lettuce, beans, peas), some herbs and herbal-flavoured liquors for their content of the 'free' anthraquinones emodin, chrysophanol and physcion. For qualitative and quantitative analysis, reversed-phase HPLC (RP-LC), gas chromatography-mass spectrometry (GC-MS) and RP-LC-MS were used. The vegetables showed a large batch-to-batch variability, from 0.04 to 3.6, 5.9 and 36 mg total anthraquinone per kg fresh weight in peas, cabbage lettuce, and beans, respectively. Physcion predominated in all vegetables. In the herbs grape vine leaves, couch grass root and plantain herb, anthraquinones were above the limit of detection. Contents ranged below 1 mg/kg (dry weight). All three anthraquinones were also found in seven of 11 herbal-flavoured liquors, in a range of 0.05 mg/kg to 7.6 mg/kg. The genotoxicity of the analysed anthraquinones was investigated in the comet assay, the micronucleus test and the mutation assay in mouse lymphoma L5178Y tk+/- cells. Emodin was genotoxic, whereas chrysophanol and physcion showed no effects. Complete vegetable extract on its own did not show any effect in the micronucleus test. A lettuce extract completely abolished the induction of micronuclei by the genotoxic anthraquinone danthron. Taking into consideration the measured concentrations of anthraquinones, estimated daily intakes, the genotoxic potency, as well as protective effects of the food matrix, the analysed constituents do not represent a high priority genotoxic risk in a balanced human diet.

Carcinogens in the diet vs. overnutrition. Individual dietary habits, malnutrition, and genetic susceptibility modify carcinogenic potency and cancer risk.

Chemical carcinogens in the diet cannot explain the cancer incidence attributed by epidemiologists to dietary factors when the calculation is based on average exposure levels and conservative estimates of carcinogenic potencies. In a previous review, the discrepancy was explained primarily by overnutrition to which a carcinogenic potency was assigned from dietary restriction experiments and the associated reduction in spontaneous tumor incidence (W.K. Lutz and J. Schlatter, Chemical carcinogens and overnutrition in diet-related cancer, Carcinogenesis 13 [1992] 2211-2216). Here, additional aspects are introduced. They focus on using individual rather than averaged data, both for exposure and susceptibility. First, under conditions of a sublinear (convex) dose-response, the cancer incidence obtained by using an average exposure level is lower than if individual exposure levels associated with particular dietary habits are taken into account. Second, carcinogenic factors, including those unrelated to the diet (e.g., smoking), can act synergistically. Third, the potency of dietary carcinogens is increased under conditions of malnutrition in the sense of a deficiency of protective factors, such as those available with fruits, vegetables, and fibers. Quantitatively, this aspect may be particularly important because it simultaneously increases the efficacy of a multitude of carcinogens. It is concluded that chemical carcinogens could be as important as overnutrition for diet-related cancer.

Threshold dose response for tumor induction by genotoxic carcinogens modeled via cell-cycle delay.

Dose-response relationships for tumor induction in animal bioassays for carcinogenicity are often postulated to include thresholds, particularly for nongenotoxic chemicals that increase the rate of cell proliferation at high doses. In this report, thresholds are postulated also for genotoxic carcinogens. The hypothesis is based on the idea of a delay of the cell cycle induced by low-level DNA damage and an acceleration at cytotoxic dose levels, thus resulting in a J-shaped (or U-shaped) dose response for cell turnover. Calculations were based on the 2-stage clonal expansion model of carcinogenesis. The background values chosen for the model parameters resulted in a 10.5% "spontaneous" 2-year cumulative tumor incidence. Using this as a starting point, a decrease by 3, 10, and 30% in the rates of cell turnover resulted in a decrease in the spontaneous tumor incidence to 9.4, 7.1 and 3.0%, respectively. Dose-responses with J-shaped curves for the rates of cell birth and death were modeled by shifted quadratic functions reaching the minimum at dose 1. Combinations with linearly increasing mutation rates also generated, under certain conditions, J-shaped dose-response curves for tumor incidence. As an example, for a 30% increase in mutation rates and a 10% decrease in cell turnover rates (both at dose 1), the dose-response curve showed an initial decrease of tumor incidence below the spontaneous rate, a reversion to the background value at 0.8 dose units, and an increase thereafter. The 0.8 dose could be considered to represent the "threshold dose." The approach presented might reconcile opposing views on thresholds on a biologically plausible mechanistic basis, and show a way for the quantitative estimation of threshold doses.

Background DNA damage for endogenous and unavoidable exogenous carcinogens: a basis for spontaneous cancer incidence?

It is undisputed today that DNA is constantly being damaged in the absence of any exposure to genotoxic carcinogens by a specific treatment or a particular habit. Damaging processes include endogenous and unavoidable exogenous sources. The list comprises chemical DNA instability (eg., depurination), spontaneous errors during DNA replication and repair, endogenous reactive chemicals (aldehydes, S-andenosylmethionine), numerous reactive oxygen species and products generated in consequence (e.g., lipid peroxides). Exogenous sources are ionizing and UV radiation, naturally occurring radioisotopes, and numerous genotoxic chemicals present in diet and air, both naturally or as contaminants. DNA repair and DNA replicative synthesis result in a constant reduction of the level of damage, but a certain steady-state level is measurable as a background at all times. The quantitative relationship between the background DNA damage and the spontaneous rates of mutation and cancer remains largely unknown. The quantitative relationship between the background DNA damange and the spontaneous rates of mutationa and caner remains largley unknown. In order to establish better correlations, more data have to be accumulated. This includes (i) measuring artefact-free levels of background DNA damage, (ii) investigating the mutagenic potency of the various lesions, (iii) including types of DNA damage other than adducts, and (iv) answer the question of organ-specific and cell type-specific +requirements for the primary DNA damage to be expressed as heritable genetic changes. The manuscripts put together for this Special Issue of Mutation Research describe the state of the art for these aspects.

Stimulation of cell division in the rat by NaCl, KCl, MgCl2, and CaCl2, and inhibition of the sodium chloride effect on the glandular stomach by ascorbic acid and beta-carotene.

Three questions associated with the stimulation of cell division by chloride salts have been investigated: (i) whether cations other than sodium show a similar effect, (ii) whether vitamins can have a preventive activity, and (iii) whether subchronic treatment with sodium chloride in the diet is also effective. Male Fischer 344 rats were given solutions of the chloride salts of sodium, potassium, magnesium, and calcium by oral gavage. Water was used for control. After 4 h, a 24-h osmotic minipump containing 5-bromo-2'-deoxyuridine was implanted subcutaneously. The forestomach and glandular stomach, as well as liver and bladder were analyzed immunohistochemically 24 h later for the proportion of cells in S phase as an indicator of the rate of replicative DNA synthesis. For both the forestomach and the glandular stomach, potassium was as potent as sodium, and the divalent cations Mg and Ca were even more potent on a molar basis. Supplementation of the diet with ascorbic acid (2 g/kg food) or beta-carotene (12.5 mg/kg food) for 1 week before gavage of the sodium chloride solution resulted in an inhibition of the stimulation of cell division. A putative tumor-chemopreventive activity of the two vitamins might therefore not only rely on their antioxidative properties but may include effects on the cell cycle. A 4-week treatment with a sodium chloride supplement in the diet (2% and 4% supplement) resulted in a significant stimulation of cell division not only in both parts of the stomach and in the bladder (with the 4% supplement) but also in the liver (even with the 2% supplement). Sodium-chloride-stimulated cell turnover therefore is a sustained effect.

Discrimination between genotoxicity and cytotoxicity for the induction of DNA double-strand breaks in cells treated with aldehydes and diepoxides.

The time-dependent dose-response relationships for the induction of DNA double-strand breaks (DSB) assessed by pulsed-field gel electrophoresis (PFGE) and for viability (evaluated by the MTT cytotoxicity test) were investigated in order to discriminate between genotoxic and cytotoxic mechanisms of DNA fragmentation. Cultured human lung epithelial cells (A549) were treated (i) with the aldehydes formaldehyde or glutaraldehyde and (ii) with the DNA-DNA interstrand crosslinkers melphalan, diepoxybutane or diepoxyoctane. Induction of DSB by formaldehyde and glutaraldehyde was seen only after cell viability was reduced to less than about 60% of the control values, indicating that DSB were the consequence of extragenomic damage and viability loss. Melphalan, diepoxybutane and diepoxyoctane induced DSB by a genotoxic mode with concentrations that did not affect cell survival: 8 h after treatment initiation both heat-labile crosslinks and DSB could be detected. Cells were not able to repair the crosslinks induced by diepoxybutane, the crosslinker with the shortest chain length. In contrast, with melphalan and diepoxyoctane, which have a longer crosslinking property considerable repair of crosslinks was observed. The molecular size distribution of the produced DNA fragments supported this mechanistic distinction. The DNA fragments generated by diepoxides were initially large, their concentration decreasing monotonously from 7 Mbp to less than 1 Mbp and were converted to smaller fragments by 72 h in the course of cell death. In contrast, DNA fragments induced by formaldehyde peaked below 1 Mbp, implicating activation of DNA-degrading enzymes.

Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts.

The quantitative relationship between DNA adducts and tumor incidence is evaluated in this review. All available data on DNA adduct levels determined after repeated administration of a carcinogen to rats or mice have been compiled. The list comprised 27 chemicals, of all major structural classes of carcinogens. For the correlation with tumor incidence, the DNA adduct levels measured at the given dose were normalized to the dose which resulted in a 50% tumor incidence under the conditions of a 2-year bioassay (TD50 dose). In rat liver, the calculated adduct concentration 'responsible' for a 50% hepatocellular tumor incidence spanned from 53 to 2083 adducts per 108 nucleotides, for aflatoxin B1, tamoxifen, IQ, MeIQx, 2,4-diaminotoluene, and dimethylnitrosamine (in this order). In mouse liver, the respective figures were 812 to 5543 adducts per 108 nucleotides, for ethylene oxide, dimethylnitrosamine, 4-aminobiphenyl, and 2-acetylaminofluorene. The observed span (40-fold in rats, 7-fold in mice) reflects differences between the various DNA adducts to lead to critical mutations. If additional carcinogens fit in with this astonishingly narrow range, the measurement of DNA adduct levels in target tissue has the potential to be not only an exposure marker but an individual cancer risk marker. For toremifen and styrene, low levels of DNA adducts were detected in rat liver at the end of a negative long-term bioassay. This shows that the limit of detection of DNA adducts can be well below the limit of detection of an increased tumor incidence. For a cancer risk assessment at low levels of DNA damage, treatment-related adducts must be discussed in relation to the background DNA damage and its inter- and intraindividual variability.

32P-Postlabeling analysis of DNA adducts of styrene 7,8-oxide at the O6-position of guanine.

A 32P-postlabeling method was established for the quantitative characterization of 2'-deoxyguanosyl O6-adducts of styrene 7,8-oxide in DNA. The two regioisomeric adducts, O6-(2-hydroxyl-1-phenylethyl)-2'-deoxyguanosine 3'-phosphate (alpha-isomer) and O6-(2-hydroxyl-2-phenylethyl)-2'-deoxyguanosine 3'-phosphate (beta-isomer), were synthesized and used for optimizing and quantifying the various analytical steps. The adducts were stable at pH 7 and 10, but not at pH 4. The adducts were sensitive to dephosphorylation during the standard nuclease P1 (NP1) treatment. Within 30 min, 73 and 94% of the alpha- and beta-isomers were digested. Adducts could not be extracted into butanol, and micropreparative chromatography on reversed-phase thin layers resulted in a loss of adducts at low levels. Therefore, further methods of enrichment had to be investigated. Micropreparative reversed-phase HPLC chromatography on a C18 column resulted in a many thousand-fold purification from the normal nucleotides. Further enrichment was achieved with a mild NP1 treatment. The phosphorylation efficiency with polynucleotide kinase was 5 and 15% for the alpha- and beta-isomers, respectively. Adduct analysis was performed with reversed-phase TLC followed by contact transfer of the origin to a polyethyleneimine-cellulose sheet and two-dimensional development. Addition of various amounts of adduct standard to the hydrolysate of 30 microg of DNA isolated from a control rat liver showed limits of detection of three and two adducts per 10(7) nucleotides for the alpha- and beta-isomers, respectively. The applicability of the newly developed method was demonstrated by the DNA analysis of styrene-exposed rats.

Dose-response relationships in chemical carcinogenesis reflect differences in individual susceptibility. Consequences for cancer risk assessment, extrapolation, and prevention.

The shape of the dose-cancer incidence curve observed in an animal bioassay for carcinogenicity of a chemical is the result of a superposition of various mechanisms contributing to the process of carcinogenesis. For genotoxic carcinogens, for instance, a sublinear (convex; up-bent) shape could be the result of a saturation of DNA repair or of a higher rate of cell turnover associated with high-dose cytotoxicity and regenerative hyperplasia. In human cancer epidemiology, the situation is more complex. The human population is very heterogeneous with respect to both genetic and life-style factors that modulate the process of tumor formation. Therefore, individuals are expected to show widely variable susceptibility to carcinogenic factors, and the dose-response curve is in fact a reflection of the tolerance distribution. Each modulating factor divides the population up into subpopulations of different susceptibility so that nonlinearities that could be present in a homogeneous population are flattened out. A linear extrapolation of a human cancer risk to low dose might therefore be appropriate under certain conditions even if the dose-response curve in animals has a strongly sigmoidal shape. For cancer prevention, the investigation of susceptibility factors is expected to be of great value. The elimination of concurrent risk factors in high-risk subpopulations or individuals might be more effective than a minor general reduction of a tolerable exposure level.

Influence of diet restriction and tumor promoter dose on cell proliferation, oxidative DNA damage and rate of papilloma appearance in the mouse skin after initiation with DMBA and promotion with TPA.

The mouse skin tumor initiation-promotion model was used to investigate the protective effect of diet restriction in mechanistic and quantitative terms. A total of five groups of 14 male NMRI mice were initiated with 100 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly with 2.5, 1.25, or 0.625 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). Food intake was ad libitum (all 3 TPA dose levels) or restricted to 70% (high and intermediate TPA dose levels). Time of appearance of the first papilloma was recorded for each mouse. Two weeks later, an osmotic minipump delivering 5-bromo-2'-deoxyuridine (BrdU) was implanted and the mouse was killed after 24 h. Cell proliferation in the epidermis was assessed by immunohistochemistry for BrdU incorporated into DNA. 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in epidermal DNA was determined by HPLC/electrochemical detection. The median latency time (t50) for the appearance of skin papilloma in the high-, intermediate-, and low-dose TPA groups fed ad libitum was 9, 15.5, and 23.5 weeks, respectively. The diet-restricted groups (high and intermediate TPA dose) showed t50 values of 16 and 26 weeks. Therefore, diet restriction to 70% had approximately the same protective effect as reducing the dose of TPA by a factor of two. Both the rate of cell proliferation and the level of 8-OH-dG in the epidermis increased with the dose of TPA. Median values were increased 3- to 4-fold at the highest dose. In controls, but not in TPA-treated animals, diet restriction resulted in a decrease for both markers, by 25 and 40% for the labeling index for cell division and the level of 8-OH-dG, respectively. Both markers showed an inverse relationship with the median papilloma latency time. On an individual basis, the correlation was significant in some groups, but only for the labeling index. The data indicate that protection from the skin tumor-promoting effect of TPA by diet restriction could be based more on a reduction of the rate of cell division than on a reduction of oxidative DNA damage.

Dose-response relationships in chemical carcinogenesis: superposition of different mechanisms of action, resulting in linear-nonlinear curves, practical thresholds, J-shapes.

The shape of a carcinogen dose-cancer incidence curve is discussed as the result of a superposition of dose-response relationships for various effects of the carcinogen on the process of carcinogenesis. Effects include direct DNA damage, e.g., by covalent binding, indirect DNA damage, e.g., by increased formation of reactive oxygen species or interaction with DNA replication or chromosome integrity. The 'fixation' of a DNA adduct as a heritable mutation depends on its pro-mutagenic potency and on the rates of DNA repair and DNA replication. Endogenous and unavoidable DNA damage is responsible for a background rate of the process of mutagenesis and carcinogenesis and forms the basis of spontaneous cancer incidence. For DNA-reactive carcinogens, linearity of the dose response at the low-dose end is expected. With increasing dose, saturation of DNA repair can introduce a sublinearity (example: dimethylnitrosamine). Stimulation of cell division as a result of high-dose toxicity and regenerative proliferation also results in a sublinear deviation from low-dose linearity. If the DNA-damaging potency of the carcinogen is low in comparison with the high-dose effects, the linear part of the low dose-cancer incidence curve might be hidden within the background variability. Under such conditions, 'practical thresholds' could be discussed (formaldehyde). If a carcinogen increases the rate of cell division or the level of oxidative stress at high dose but has an antimitogenic or antioxidative effect at low dose, a J-shaped (or: U-shaped) curve with a decrease of the spontaneous tumor incidence at low dose could result (caffeic acid; TCDD). This phenomenon has been observed even under conditions of a genotoxic contribution (ionizing radiation; diesel exhaust particles). For a mechanism-based assessment of a low-dose cancer risk, information on the various modes of action and modulations should be available over the full dose range, and models should be refined to incorporate the respective information.

Factors affecting the genotoxic potency ranking of natural anthraquinones in mammalian cell culture systems.

We had reported that the plant-derived 1,8-dihydroxyanthraquinone derivatives, emodin and danthron, were clearly genotoxic in mouse lymphoma L5178Y cells, whereas chrysophanol was only weakly genotoxic and physcion not at all. Danthron was more potent than emodin. Furthermore, we had found that these compounds bound non-covalently to DNA and inhibited topoisomerase II activity. Interestingly, in these systems emodin was more potent than danthron. This inversion of the ranking prompted us to investigate the underlying mechanism. Since emodin shows a high serum-protein binding affinity, horse serum used as a media-supplement in the mouse lymphoma genotoxicity assays was analyzed for a potential selective scavenging of emodin. Non-covalent DNA-binding in mouse lymphoma L5178Y cells was investigated in the absence or presence of serum. In the presence of 10% serum, the DNA-binding potency of emodin was markedly reduced and was lower than that of danthron. We also applied mutation assays with mouse lymphoma cells and AS52 cells and varied the serum concentration used. In the absence of serum emodin showed slightly higher mutagenicity in AS52 cells than danthron. At reduced serum concentration (0.5%) emodin was strongly cytotoxic to the mouse lymphoma cells. For chrysophanol and physcion, a considerable reduction of the non-covalent DNA-binding potency in intact cells was found when compared to danthron, in concordance with their lower genotoxic potency. Overall, these data support the understanding that the genotoxicity of anthraquinones is, at least in part, mediated by non-covalent DNA-binding.