Investigation of the potential for binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA in vivo.

It was the aim of this investigation to determine whether or not covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of rodents with high doses of DEHP. DEHP radiolabeled in different positions was administered orally to female F344 rats with or without pretreatment for 4 weeks with 1% unlabeled DEHP in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. Administration of [14C]carboxylate-labeled DEHP resulted in no measurable DNA radioactivity. With DEHP [14C]- and [3H]-labeled in the alcohol moiety as well as with 2-ethyl[1-14C]hexanol, radioactivity was clearly measurable in the DNA. HPLC analysis of enzyme-degraded DNA revealed that the normal nucleosides had incorporated radiolabel whereas no radioactivity was detectable in those fractions where the carcinogen-modified nucleoside adducts are expected. A quantitative evaluation of the negative data in terms of a limit of detection for a covalent binding index (CBI) indicates that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP in rodents

Tracing Back the Eighteenth Century "Nuptiality Transition" in Finland

Official Finnish population statistics starting in 1751 do not provide cross-classifications of age and marital status before 1880. However, declining overall proportions married and declining fertility rates suggest a major change in the marriage pattern during the second half of the eighteenth century. Five sources of information, namely overall proportions married, annual numbers of marriages, sizes of marriage cohorts relative to mortality-adjusted birth cohorts, information on the modal age at marriage from lagged correlation analysis between sizes of birth and marriage cohorts, and trends in age-specific fertility are analyzed to provide information on nuptiality trends. A simulation model based on the Coale-McNeill marriage model yields parameter estimates for the Finnish provinces in 1751-1772, which allow calculation of the "Princeton Index" I_m. The results suggest a "nuptiality transition" from early and almost universal marriage to the so-called "European Marriage Pattern", which is characterized by late marriage and high proportions of unmarried. Provincial level analysis reveals significant east-west differentials with higher proportions marrying and lower mean ages at marriage in the eastern parts of Finland. Non-quantitative historical evidence in general also supports the assumption of a major change in the Finnish nuptiality pattern during the second half of the eighteenth century

Dose-Incidence Relationships Derived from Superposition of Distributions of Individual Susceptibility on Mechanism-Based Dose Responses for Biological Effects

Dose-response relationships for incidence are based on quantal response measures. A defined effect is either present or not present in an individual. The dose-incidence curve therefore reflects differences in individual susceptibility (the "tolerance distribution”). At low dose, only the more susceptible individuals manifest the effect, while higher doses are required for more resistant individuals to be recruited into the affected fraction of the group. Here, we analyze how such dose-incidence relationships are related to mechanism-based dose-response relationships for biological effects described on a continuous scale. As an example, we use the quantal effect "cell division” triggered by occupancy of growth factor receptors (R) by a hormone or mitogenic ligand (L). The biologically effective dose (BED) is receptor occupancy (RL). The dose-BED relationship is described by the hyperbolic Michaelis-Menten function, RL/Rtot = L / (L + KD). For the conversion of the dose-BED relationship to a dose-cell division relationship, the dose-BED curve has to be combined with a function that describes the distribution of susceptibilities among the cells to be triggered into mitosis. We assumed a symmetrical sigmoid curve for this function, approximated by a truncated normal distribution. Because of the supralinear dose-BED relationship due to the asymptotic saturation of the Michaelis-Menten function, the composite curve that describes cell division (incidence) as a function of dose becomes skewed to the right. Logarithmic transformation of the dose axis reverses this skewing and provides a nearly perfect fit to a normal distribution in the central 95% incidence range. This observation may explain why dose-incidence relationships can often be described by a cumulative normal curve using the logarithm of the administered dose. The dominant role of the tolerance distribution for dose-incidence relationships is also illustrated with the example of a linear dose-BED relationship, using adducts to protein or DNA as the BED. Superimposed by a sigmoid distribution of individual susceptibilities, a sigmoid dose-incidence curve results. Linearity is no longer observed. We conclude that differences in susceptibility should always be considered for toxicological risk assessment and extrapolation to low dos

Methylation of DNA by incubation with methylamine and nitrite

DNA was incubated in septum-closed reaction vials with [14C]methylamine and nitrite. The DNA was purified, hydrolysed with hydrochloric acid, and the purines were analysed by h.p.I.c. 7-Methylguanine was detectable as a result of DNA methylation in experiments performed in 100 mM acetate at pH 4. Using different concentrations of amine and nitrite a first order reaction for total amine and a second order for total nitrite could be shown. A study on the pH dependence using 100 mM malonate buffer, pH 2.0-6.0, revealed a maximum rate at pH 3.5, with steep slopes above and below this pH value, in agreement with a mathematical analysis of the reaction equations. The data show that the alkylating agent formed spontaneously by nitrosation and deamination of a primary amine has a long enough lifetime to react with DNA in vitro. Using the reaction orders established here, an extrapolation to lower concentrations found in the stomach can now be performed. Future in vivo experiments on the methylation of gastro-intestinal DNA then would show to what extent DNA in a cell is protected from alkylatio

Covalent binding of [2-14C]2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) to mouse DNA in vivo

Female BALB/c mice were administered intragastrically with equimolar amounts of either [2-14C]2-amino-3,8-dimethyl-[4,5-f]quinoxaline (MeIQx) or 2-acetylamino[9-14C]fluorene (2AAF). DNA was isolated from tissues of mice killed either 6 or 24 h after administration. Analysis of liver DNA nucleotide digests by HPLC analysis revealed that all of the radioactivity was attributable to adduct formation. The specific activities of DNA samples were converted to covalent binding indices (CBI, μmol adduct per mol DNA nucleotides/mmol chemical applied per kg animal body weight). CBI values of 25 and 9 were determined for 2AAF and MeIQx in the livers of mice killed 6 h after dosing. The values were in general agreement with the moderate carcinogenic potency of these compounds. The specific activities of DNA preparations obtained from the kidneys, spleens, stomachs, small intestines and large intestines of mice treated with MeIQx and killed 6 h after dosing were 5- to 35-times less than those obtained with the liver. DNA isolated from the lungs (a target organ for MeIQx tumorigenicity) of MeIQx-treated mice was not radiolabelled at the limit of detection (CBI <0.3). With the exception of the gastrointestinal tract, the specific activities of DNA samples isolated from mice killed 6 h after administration were higher than those from mice killed after 24

Short communication : Mouse skin papilloma formation by chronic dermal application of 7,12-dimethylbenz[a]anthracene is not reduced by diet restriction

No abstract availabl

Different Types of Combination Effects for the Induction of Micronuclei in Mouse Lymphoma Cells by Binary Mixtures of the Genotoxic Agents MMS, MNU, and Genistein

Distinction between dose addition and response addition for the analysis of the toxicity of mixtures may allow differentiation of the components regarding similar versus independent mode of action. For nonlinear dose responses for the components, curves of dose addition and response addition differ and embrace an "envelope of additivity.” Synergistic or antagonistic interaction may then be postulated only if the mixture effect is outside this surface. This situation was analyzed for the induction of micronuclei in L5178Y mouse lymphoma cells by the two methylating agents methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU) and the topoisomerase-II inhibitor genistein (GEN). All three chemicals reproducibly generated sublinear (upward convex) dose-response relationships. For the analysis of mixture effects, these genotoxic agents were investigated in the three binary combinations. Statistical testing for dose addition along parallel exponential dose responses was performed by linear regression with interaction based on the logarithm of the number of cells that contain micronuclei. For MMS+MNU, the mixture effect was compatible with dose addition (i.e., significantly larger than calculated for the addition of net responses). For MMS+GEN, the measured effect was larger than for response addition but smaller than for dose addition. For MNU+GEN, the measured effect was below response addition, indicative of true antagonism. In the absence of knowledge on the sublinear dose-response relationships for the individual components, a synergistic effect of MMS on both MNU and GEN would have been postulated erroneously. The observed difference between MMS and MNU when combined with GEN would not have been predicted on the basis of a simplistic interpretation of DNA methylation as the mode of action and may be due to differences in the profile of DNA methylations and/or epigenetic effects. We conclude that knowledge of nonlinearities of the dose-response curves of individual components of a mixture can be crucial to analyze for synergism or antagonism and that an in-depth mechanistic knowledge is useful for a prediction of similarity or independence of actio

Dose-Incidence Modeling: Consequences of Linking Quantal Measures of Response to Depletion of Critical Tissue Targets

In developing mechanistic PK-PD models, incidence of toxic responses in a population has to be described in relation to measures of biologically effective dose (BED). We have developed a simple dose-incidence model that links incidence with BED for compounds that cause toxicity by depleting critical cellular target molecules. The BED in this model was the proportion of target molecule adducted by the dose of toxic compound. Our modeling approach first estimated the proportion depleted for each dose and then calculated the tolerance distribution for toxicity in relation to either administered dose or log of administered dose. We first examined cases where the mean of the tolerance distribution for toxicity occurred when a significant proportion of target had been adducted (i.e., more than half). When a normal distribution was assumed to exist for the relationship of incidence and BED, the tolerance distribution based on administered dose for these cases becomes asymmetrical and logarithmic transformations of the administered dose axis lead to a more symmetrical distribution. These linked PK-PD models for tissue reactivity, consistent with conclusions from other work for receptor binding models (Lutz et al., 2005), indicate that log normal distributions with administered dose may arise from normal distributions for BED and nonlinear kinetics between BED and administered dose. These conclusions are important for developing biologically based dose response (BBDR) models that link incidences of toxicity or other biological responses to measures of BE