An integrated QSAR-PBK/D modelling approach for predicting detoxification and DNA adduct formation of 18 acyclic food-borne α,ß-unsaturated aldehydes.

Acyclic α,ß-unsaturated aldehydes present in food raise a concern because the α,ß-unsaturated aldehyde moiety is considered a structural alert for genotoxicity. However, controversy remains on whether in vivo at realistic dietary exposure DNA adduct formation is significant. The aim of the present study was to develop physiologically based kinetic/dynamic (PBK/D) models to examine dose-dependent detoxification and DNA adduct formation of a group of 18 food-borne acyclic α,ß-unsaturated aldehydes without 2- or 3-alkylation, and with no more than one conjugated double bond. Parameters for the PBK/D models were obtained using quantitative structure-activity relationships (QSARs) defined with a training set of six selected aldehydes. Using the QSARs, PBK/D models for the other 12 aldehydes were defined. Results revealed that DNA adduct formation in the liver increases with decreasing bulkiness of the molecule especially due to less efficient detoxification. 2-Propenal (acrolein) was identified to induce the highest DNA adduct levels. At realistic dietary intake, the predicted DNA adduct levels for all aldehydes were two orders of magnitude lower than endogenous background levels observed in disease free human liver, suggesting that for all 18 aldehydes DNA adduct formation is negligible at the relevant levels of dietary intake. The present study provides a proof of principle for the use of QSAR-based PBK/D modelling to facilitate group evaluations and read-across in risk assessment.

A physiologically based in silico model for trans-2-hexenal detoxification and DNA adduct formation in human including interindividual variation indicates efficient detoxification and a negligible genotoxicity risk.

A number of α,ß-unsaturated aldehydes are present in food both as natural constituents and as flavouring agents. Their reaction with DNA due to their electrophilic α,ß-unsaturated aldehyde moiety may result in genotoxicity as observed in some in vitro models, thereby raising a safety concern. A question that remains is whether in vivo detoxification would be efficient enough to prevent DNA adduct formation and genotoxicity. In this study, a human physiologically based kinetic/dynamic (PBK/D) model of trans-2-hexenal (2-hexenal), a selected model α,ß-unsaturated aldehyde, was developed to examine dose-dependent detoxification and DNA adduct formation in humans upon dietary exposure. The kinetic model parameters for detoxification were quantified using relevant pooled human tissue fractions as well as tissue fractions from 11 different individual subjects. In addition, a Monte Carlo simulation was performed so that the impact of interindividual variation in 2-hexenal detoxification on the DNA adduct formation in the population as a whole could be examined. The PBK/D model revealed that DNA adduct formation due to 2-hexenal exposure was 0.039 adducts/108 nucleotides (nt) at the estimated average 2-hexenal dietary intake (0.04 mg 2-hexenal/kg bw) and 0.18 adducts/108 nt at the 95th percentile of the dietary intake (0.178 mg 2-hexenal/kg bw) in the most sensitive people. These levels are three orders of magnitude lower than natural background DNA adduct levels that have been reported in disease-free humans (6.8-110 adducts/108 nt), suggesting that the genotoxicity risk for the human population at realistic dietary daily intakes of 2-hexenal may be negligible.

Genotoxic effects in the Eastern mudminnow (Umbra pygmaea) after prolonged exposure to River Rhine water, as assessed by use of the in vivo SCE and Comet assays.

The production of drinking water from river water requires a certain minimal river water quality. The Association of River Rhine Water Works (RIWA), therefore, operates a monitoring network. In vitro mutagenicity studies have shown that the genotoxicity of the River Rhine water steadily decreased from 1981 until 2001. Compared to a study in 1978, a decrease in genotoxicity was also observed in an in vivo genotoxicity study in 2005, in which Eastern mudminnows (Umbra pygmaea) were exposed to River Rhine water, and gill cells were used for the Sister Chromatid Exchange (SCE) test and the Comet assay. In this 2005 study, the in vivo genotoxicity increased upon extending exposure of the fish from 3 to 11 days. Therefore, the objectives of this study were to investigate (i) whether new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978, (ii) whether the Comet assay is a suitable alternative to the SCE assay, and (iii) whether further prolonged exposure results in a further increase in in vivo genotoxicity. The new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978. The Comet assay is a useful addition but does not provide a substitute for the SCE endpoint in these in vivo genotoxicity studies. Prolonging the exposure time of Eastern mudminnows to River Rhine water from 11 to 42 days did not give a significant increase in SCEs and DNA damage (Comet assay) in gill cells.

Physiologically based biokinetic (PBBK) model for safrole bioactivation and detoxification in rats.

A physiologically based biokinetic (PBBK) model for alkenylbenzene safrole in rats was developed using in vitro metabolic parameters determined using relevant tissue fractions. The performance of the model was evaluated by comparison of the predicted levels of 1,2-dihydroxy-4-allylbenzene and 1'-hydroxysafrole glucuronide to levels of these metabolites reported in the literature to be excreted in the urine of rats exposed to safrole and by comparison of the predicted amount of total urinary safrole metabolites to the reported levels of safrole metabolites in the urine of safrole exposed rats. These comparisons revealed that the predictions adequately match observed experimental values. Next, the model was used to predict the relative extent of bioactivation and detoxification of safrole at different oral doses. At low as well as high doses, P450 mediated oxidation of safrole mainly occurs in the liver in which 1,2-dihydroxy-4-allylbenzene was predicted to be the major P450 metabolite of safrole. A dose dependent shift in P450 mediated oxidation leading to a relative increase in bioactivation at high doses was not observed. Comparison of the results obtained for safrole with the results previously obtained with PBBK models for the related alkenylbenzenes estragole and methyleugenol revealed that the overall differences in bioactivation of the three alkenylbenzenes to their ultimate carcinogenic 1'-sulfooxy metabolites are limited. This is in line with the generally less than 4-fold difference in their level of DNA binding in in vitro and in vivo studies and their almost similar BMDL(10) values (lower confidence limit of the benchmark dose that gives 10% increase in tumor incidence over background level) obtained in in vivo carcinogenicity studies. It is concluded that in spite of differences in the rates of specific metabolic conversions, overall the levels of bioactivation of the three alkenylbenzenes are comparable which is in line with their comparable carcinogenic potential.

Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect.

Estragole is a natural constituent of several herbs and spices including sweet basil. In rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1'-sulfooxyestragole resulting in DNA adduct formation. The present paper identifies nevadensin as a basil constituent able to inhibit DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1'-hydroxyestragole and nevadensin. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1'-hydroxyestragole. The Ki for SULT inhibition by nevadensin was 4 nM in male rat and human liver fractions. Furthermore, nevadensin up to 20 microM did not inhibit 1'-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the recently developed physiologically based biokinetic (PBBK) rat and human models for estragole bioactivation and detoxification. The results predict that co-administration of estragole at a level inducing hepatic tumors in vivo (50mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1'-sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition would still amount to more than 83%. Altogether these data point at a nevadensin-mediated inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing the capacity to detoxify 1'-hydroxyestragole via glucuronidation or oxidation. These data also point at a potential reduction of the cancer risk when estragole exposure occurs within a food matrix containing SULT inhibitors compared to what is observed upon exposure to pure estragole.

Genotoxic effects in the Eastern mudminnow (Umbra pygmaea L.) after exposure to Rhine water, as assessed by use of the SCE and Comet assays: a comparison between 1978 and 2005.

Surface water used for drinking-water preparation requires continuous monitoring for the presence of toxic compounds. For monitoring of genotoxic compounds fish models have been developed, such as the Eastern mudminnow (Umbra pygmaea L.) because of its clearly visible 22 meta-centric chromosomes. It was demonstrated in the late seventies that Rhine water was able to induce chromosome aberrations and sister chromatid exchange in this fish species. Although in vitro mutagenicity studies of the RIWA (Rhine Water Works, The Netherlands) have shown that the genotoxicity of the river Rhine steadily decreased during the last decades, there is still concern about the presence of some residual mutagenicity. In addition, in most studies the water samples have been tested only in in vitro test systems such as the Salmonella-microsome test. For this reason, and in order to be able to make a comparison with the water quality 27 years ago, a study was performed with the same experimental design as before in order to measure the effect of Rhine water on the induction of SCE in the Eastern mudminnow. As a new test system the single cell gel electrophoresis assay (Comet assay) was performed. Fish were exposed to Rhine water or to groundwater for 3 and 11 days in flow-through aquaria. Fish exposed for 11 days to Rhine water had a significantly higher number of SCE and an increased comet tail-length compared with control fish exposed to groundwater. After exposure for three days to Rhine water there was no difference in SCE and a slightly increased comet tail-length compared with the control. It was concluded that genotoxins are still present in the river Rhine, but that the genotoxic potential has markedly decreased compared with 27 years ago. Furthermore, the Comet assay appears to be a sensitive assay to measure the genotoxic potential of surface waters in fish.

In vitro biomonitoring in polar extracts of solid phase matrices reveals the presence of unknown compounds with estrogenic activity.

Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro 'estrogen receptor-mediated chemical activated luciferase gene expression' (ER-CALUX) assay was applied to samples from various locations in the Netherlands. Estrogenic activity measured in polar fractions of particulate matter and sediment extracts ranged from below detection limit to up to 4.5 pmol estradiol equivalents (EEQ)/g dry weight. Estrogenic activity in freshwater river sediments was up to five times higher compared to sediments from large lakes and coastal locations. Tissue extracts EEQs were determined in bream (Abramis brama), flounder (Platichthysflesus), freshwater mussels (Dreissena polymorpha) and marine mussels (Mytilus edulis). The highest biota EEQ levels were found in the freshwater zebra mussel (30 pmol EEQ/g lipid). One sample site showed greatly elevated EEQs in sediment and biota, which correlated with effects found in the wild populations of bream. The EEQ activity of the unknown compounds in the polar fraction mostly was much higher than the calculated EEQ levels based on known estrogens in the non-polar fraction (previously published data).

Delayed effects of pre- and early-life time exposure to polychlorinated biphenyls on tadpoles of two amphibian species (Xenopus laevis and Rana temporaria).

This study examined the effects of polychlorinated biphenyls (PCBs) on the development of amphibians using Xenopus laevis and Rana temporaria as experimental animals. Amphibians were exposed at different life stages and via different routes to the technical mixtures Clophen A50 and Aroclor 1254 or to a non-ortho PCB congener (PCB 126). The effects of PCB exposure in amphibians, such as mortality, number and pattern of malformations, or body weight at the end of successful metamorphosis of tadpoles, depends on the route, the point of time of exposure during the complex life cycle of amphibians, and the length of the observation period. Retinoid concentrations were significantly altered in PCB dosed embryos. Presently used early-life time test systems such as the FETAX assay may underestimate toxic effects of compounds with long term response such as PCBs on amphibians.

Transport and metabolism of glutathione conjugates of menadione and ethacrynic acid in confluent monolayers of rat renal proximal tubular cells.

Confluent monolayers of primary rat renal proximal tubular (RPT) cells were used to compare transepithelial transport and concomitant metabolism of two different glutathione (GSH) S-conjugates. For the GSH-conjugated quinone compound, [35S]GSH-conjugated menadione (MGNQ), no specific transepithelial transport was observed. Most likely, [35S]MGNQ passed the monolayer via paracellular leakage as the result of a reduction in monolayer integrity due to toxicity via extensive redox cycling of the quinone under the culture conditions. RPT cell monolayers metabolise MGNQ into a cysteinylglycine conjugate, which after intramolecular cyclization yields 2H-(3-glycinyl)-9-hydroxy-10-methyl-1,4-naphthothiazine. Acivicin, an inhibitor of gamma-glutamyltranspeptidase, inhibited the formation of this 1,4-napthothiazine adduct. The second product formed is 1,4-napthothiazine formed by loss of glycine via the action of dipeptidases. Similarly, no basolateral (B) to apical (A) transport of a GSH-conjugated alpha, beta unsaturated ketone, [14C]ethacrynic acid (EASG), occurred. However, net transport of [14C] radioactivity could be observed from A=>B direction. After 8 h, 23% of total [14C] radioactivity was transported from the apical to the basolateral chamber. In both the apical and basolateral chambers, free, unconjugated ethacrynic acid (EA) was observed. gamma GT-mediated metabolism of EASG to the much more unstable cysteinylglycine conjugate leads to relatively large amounts of free EA. Thus, the GSH conjugate is not transported but rather the cysteine adduct and/or free, unconjugated EA. In agreement with this, acivicin reduced A=>B transport of EASG and inhibited the formation of free EA. In conclusion, the confluent monolayers of RPT cells do not or no longer possess active basolateral transport systems for GSH conjugates. However, they are still quite useful for studying biotransformation reactions of thioether conjugates.

Inhibition of binding of thyroxin to transthyretin by outdoor and indoor airborne particulate matter and effects on thyroid hormone and vitamin A metabolism in rats.

Previous studies have indicated that chemicals, e.g., hydroxylated polychlorinated biphenyls (PCBs) decrease plasma thyroxin levels through interaction with transthyretin. As a consequence, plasma vitamin A and thyroxin are depleted. Both thyroxin and vitamin A are important regulators of normal epithelial differentiation and proliferation and might act in the promotion phase of carcinogenesis. In the present report, extracts of indoor and outdoor airborne particulate matter (APM), originating from different pollution sources, were tested for a possible interaction in vitro and in vivo with thyroxin and vitamin A. Results indicated that all APM extracts significantly interfered with thyroxin binding to transthyretin. In all cases, the more polluted samples inhibited binding more than the corresponding control samples. Exposure of rats to a cigarette smoke extract induced clear changes in plasma thyroxin and triiodothyronin hormone levels. Triiodothyronin levels were reduced most. Effects on plasma vitamin A levels were also found, but these were less clear. In addition a decrease in lung vitamin A levels was found, but this decrease was not statistically significant. Exposure of rats to an outdoor APM extract induced significant changes in plasma triiodothyronin levels. Effects on thyroxin levels were not significant, although the same trend was seen as for the cigarette smoke APM extract. No changes in plasma or liver vitamin A concentrations were induced by the outdoor APM extract. Lung vitamin A levels were decreased, but again this decrease was not statistically significant. Taken together, the present report shows that APM extracts have the potency to interfere with thyroid hormone metabolism both in vitro and in vivo and probably with vitamin A metabolism as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of raising and lowering intracellular glutathione levels on the cytotoxicity of allyl isothiocyanate, tert-butylhydroperoxide and chlorodinitrobenzene.

The influence of intracellular glutathione (GSH) on cytotoxicity was studied in vitro by altering the intracellular GSH level of Chang liver cells. Lowering the GSH level was achieved by treating cells with diethyl maleate (155 mum), while the intracellular GSH level was raised by cellular incubation with the monoethyl ester of glutathione (20 mm). The cytoxicity of three compounds that are known to react differently with GSH was tested using the cloning efficiency assay. Allyl isothiocyanate reacts reversibly with GSH and in previous work it was shown that the toxicity of the GSH conjugate was due to the release of the free isothiocyanate. In this study we found that changing the intracellular GSH level had no influence on the cytotoxicity of this compound. This points to the cell membrane as the primary target, although the possibility of GSH being involved as a transporting agent cannot be completely excluded. Chlorodinitrobenzene conjugates easily with GSH, and in bacterial assays it was found that depletion of intracellular GSH enhanced the mutagenic effect. After raising the intracellular GSH level in Chang cells this compound was found to be more toxic, and after lowering the GSH level less toxicity was found, indicating a role for GSH in the activation of chlorodinitrobenzene. For comparison tert-butylhydroperoxide was chosen, because it is known to be detoxified by GSH. Lowering the intracellular GSH level did indeed result in higher cytotoxicity, but after elevating the GSH level no increase in cloning efficiency was observed, indicating that there are more factors involved in tert-butylhydroperoxide cytotoxicity.