[Folic acid fortification: prevention as well as promotion of cancer]. / Foliumzuurverrijking: zowel preventie als bevordering van kanker.

In many countries foods are fortified with folic acid to prevent neural-tube defects. Beneficial effects on cancer, cardiovascular diseases and dementia are also assumed. Fortification with folic acid is not allowed in The Netherlands, although exemption can be granted. As well as beneficial effects, harmful effects may also occur. In addition to masking vitamin-B12 deficiency, there is some evidence that folic acid may promote progression of established tumours in laboratory animals and humans. In addition, it has been hypothesized that fortification with folic acid may have further negative effects on cancer through genetic selection. Given the high prevalence of cancer, these potentially harmful effects should also be taken into account in the Dutch debate on the advantages and disadvantages of folic acid fortification.

Effect of bile type on the bioaccessibility of soil contaminants in an in vitro digestion model.

Soil ingestion is an important pathway of exposure for many nonvolatile contaminants for man and in particular for children. A fraction of the ingested contaminant may not dissociate from the soil particles during digestion in the gastrointestinal tract, and is thus not available for transport across the intestinal epithelium. In order to estimate the contaminant fraction that is mobilized from soil, i.e., the bioaccessible fraction, several in vitro digestion models have been developed. The currently existing digestion models display many differences. One aspect that may affect bioaccessibility and may induce differences between digestion models is the bile that is used. Often freeze-dried bile of animal origin is preferred to purified bile salts. However, also the animal origin of bile may give rise to differences in bioaccessibility because bile composition appears to be species dependent. In the present study, we compared the bioaccessibility of benzo[a]pyrene, arsenic, cadmium, and lead of four different soils after digestion with ox bile from two different suppliers, pig bile, and chicken bile. Bioaccessibility appeared to vary amongst the different soils and contaminants. Only chicken bile increased the bioaccessibility of lead and cadmium significantly and relevantly for one of four soils. For chicken bile, the bioaccessibility of lead was 3-5.5 times greater than for the other bile types and the bioaccessibility of cadmium was 1.5 times greater. In all other cases, the bioaccessibility differences were less than 10%, which is considered irrelevant for risk assessment purposes.

Development of an in vitro digestion model for estimating the bioaccessibility of soil contaminants.

Soil ingestion can be a major route of human exposure to many immobile soil contaminants. The present risk assessment is based on toxicity studies in which contaminants are typically ingested in liquid or food matrices. The difference in bioavailability of contaminants ingested in a soil matrix is not taken into account. To become bioavailable, contaminants first need to become bioaccessible, i.e., they must be mobilized from the soil during digestion. Soil contaminants may be less bioaccessible than contaminants from liquid or food, so that the risks can be overestimated. This article describes the development of an in vitro human digestion model that is physiologically based. It can be used as a tool to assess bioaccessibility. We explain the rationale behind the experimental design of the model. We address the aspects of the simulated compartments of the gastrointestinal tract, temperature, soil-to-fluid ratio, ratio of digestive juices, transit times, centrifugation, pH values, mixing, constituents and their concentrations, and bile. The optimized in vitro digestion model was applied in a case study. The bioaccessibility of lead in pottery flakes with glazing was determined and compared to the bioaccessibility of lead in the soil from which the pottery flakes were removed. The data indicate that pottery flake lead is considerably less bioaccessible (0.3 +/- 0.2%) than lead in soil without pottery flakes (42-66% at the same site, and 28-73% at other sites in the same town). Furthermore, bioaccessibility values of lead in soil appear to be less than calculated bioaccessibility values for dietary lead (which are based on the criterion used by the Dutch risk assessment and on literature absorption data). This indicates that accounting for the matrix of ingestion can affect the exposure assessment for lead. The in vitro digestion model is a promising tool for studying the effect of the ingestion matrix on bioaccessibility.

Comparison of in vitro and in vivo developmental toxicity and pharmacokinetics of phenytoin in the rat.

The rat whole embryo culture was compared to an in vivo experiment with regard to embryotoxicity as well as exposure characteristics, using phenytoin as a model compound. Intra-embryonic concentrations and their embryotoxic effects were determined on gestation day 11 after in vitro administration of 50-150 microg/ml or in vivo gavage of 500-1500 mg/kg body-weight on gestation day 10. In addition, exposure kinetics were studied in vivo after a single oral dose on gestation day 10, and developmental defects on gestation day 21 were scored. The embryotoxic effects observed on gestation day 11 were more pronounced after in vitro exposure in comparison to in vivo exposure at similar intra-embryonic concentrations. Exposure of phenytoin on gestation day 10 in vitro via the culture medium resulted in general embryotoxicity on gestation day 11, whereas in vivo effects as determined on gestation day 11 were minimal. Plasma concentrations of phenytoin increased and plateaued around 35 microg/ml during the 48 hr monitoring period. Plasma concentration curves and pharmacokinetic parameters did not show remarkable differences between the dose groups, indicating that absorption is the limiting factor at the dose range used. Although the developmental effects were minimal as observed in vivo on gestation day 11, specific malformations (defects encompassing the urogenital. craniofacial and skeletal systems) were observed on gestation day 21. These findings show that with similar intra-embryonic concentrations of phenytoin the embryotoxicity in rat whole embryo culture was not comparable with the in vivo embryotoxicity as determined on gestation day 11. This discrepancy may at least partly be explained by differences in exposure characteristics.

Do dietary phytochemicals with cytochrome P-450 enzyme-inducing activity increase high-density-lipoprotein concentrations in humans?

Low plasma concentrations of high-density lipoprotein (HDL) are associated with increased risk of coronary heart disease. Several drugs that induce the microsomal cytochrome P-450-dependent enzyme system in liver and intestine, the sites of HDL apolipoprotein (apo) A-I and A-II synthesis, raise plasma HDL concentrations in humans. To test the hypothesis that phytochemicals with cytochrome P-450-inducing activity may also increase plasma HDL concentrations, two controlled dietary trials were undertaken in healthy nonsmoking males aged 20-28 y. One study examined the effect of replacing 300 g glucosinolate-free vegetables with 300 g Brussels sprouts/d for 3 wk. The other study examined the effects of 150 mg eugenol/d in capsule form, using a double-blind, placebo-controlled crossover design. There were no significant increases in plasma apo A-I, apo A-II, HDL cholesterol, or HDL phospholipids. These results suggest that dietary phytochemicals that induce members of the cytochrome P-450 system do not necessarily raise plasma HDL concentrations in humans, but do not exclude the possibility that some phytochemicals may have such an effect.

Effect of eugenol on the mutagenicity of benzo[a]pyrene and the formation of benzo[a]pyrene-DNA adducts in the lambda-lacZ-transgenic mouse.

To study the possible reduction by eugenol of the mutagenicity and genotoxicity of benzo[a]pyrene (B[a]P) in vivo, the lambda-lacZ-transgenic mouse strain 40.6 (Muta Mouse) was used. Male mice were fed a diet containing 0.4% (w/w) eugenol or a control diet for 58 days. On day 10, half of the mice received an i.p. dose of 100 mg/kg b.w. B[a]P. The lacZ mutants were recovered by packaging of DNA isolated from liver into lambda phage, and expressed in E. coli C lacZ-recA-galE- bacteria. In both control mice and mice fed the eugenol diet, B[a]P treatment resulted in a similar, significant increase in lacZ mutant frequency. Eugenol was not mutagenic by itself. By 32P-postlabelling analysis of the liver DNA using an analysis method with chromatographic conditions for B[a]P-DNA adducts, no effect of eugenol on the formation of B[a]P-DNA adducts in the lambda-lacZ-transgenic mouse was found. By 32P-postlabelling analysis using an alkenylbenzene solvent system the amount of B[a]P-DNA adducts was lower in mice fed the eugenol diet than in mice fed the control diet but the decrease was not statistically significant. However, one spot indicative of an eugenol-associated DNA adduct was detected. The present data provide no evidence for antimutagenic or antigenotoxic potential of eugenol in vivo. Furthermore, they suggest genotoxicity in vivo of eugenol per se.

Effect of short-term dietary administration of eugenol in humans.

1. In order to study the antigenotoxic potential of eugenol in humans, ten healthy non-smoking males ingested a daily amount of 150 mg eugenol or the placebo for seven consecutive days. After a washout period of one week, groups ingesting eugenol or the placebo were crossed and received the other treatment for seven consecutive days. 2. On days 8 and 22 blood samples were taken for the assessment of standard clinical biochemical parameters. To study the possible antigenotoxic effect of eugenol, on day 8 and 22 blood samples were collected and exposed in vitro to the established genotoxic agents mitomycin C and vinblastine. After exposure the percentage of cells with chromosome aberrations and micronuclei was determined in cultured white blood cells. On days 8 and 22 paracetamol (500 mg p.o.) was administered as test substance to measure phase-II biotransformation capacity. Glutathione-S-transferase (GST) activities were determined in erythrocytes and blood plasma. 3. No significant differences in the clinical biochemical parameters were detected between the eugenol-period and the placebo-period, indicating that daily administration of 150 mg eugenol for 7 days has no toxic affects. 4. No significant differences on the cytogenetic parameters were found after ingestion of eugenol. Thus, there are no indications for an antigenotoxic potential of eugenol in humans, consuming daily 150 mg eugenol for 7 days. 5. A significant reduction in alpha-class GSTs in plasma (P < 0.05), but not in the other measured biotransformation parameters, was found in volunteers during the eugenol-periods as compared to the placebo-period. This may either reflect GST-inhibition by eugenol or protection against background damage of liver cells by eugenol.

Inhibition of rat, mouse, and human glutathione S-transferase by eugenol and its oxidation products.

The irreversible and reversible inhibition of glutathione S-transferases (GSTs) by eugenol was studied in rat, mouse and man. Using liver cytosol of human, rat and mouse, species differences were found in the rate of irreversible inhibition of GSTs by eugenol in the presence of the enzyme tyrosinase. Tyrosinase was used to oxidize eugenol. No inhibition was observed in the absence of tyrosinase. The rate of irreversible inhibition of GSTs was highest in mouse cytosol, and lowest in rat cytosol. In addition, the irreversible inhibition of human and rat GSTs by eugenol was studied using purified isoenzymes of man and rat. The human GST isoenzymes A1-1, M1a-1a and P1-1 and the rat GST isoenzymes 1-1, 2-2, 3-3, 4-4 and 7-7 were irreversibly inhibited by eugenol in the presence of tyrosinase. In this respect human GST P1-1 and rat GST 7-7 were by far the most sensitive enzymes; human GST A2-2 was not inhibited. Indications were found that human GST P1-1 may be inhibited via three mechanisms: in addition to the well documentated nucleophilic addition of quinones and oxidation of cysteine residues, a covalent subunit cross-linking was also observed. The reversible inhibition of human and rat GST by eugenol, eugenol methyl ether, isoeugenol methyl ether, 2-allylphenol and 4-propylphenol was also studied using purified isoenzymes. The reversible inhibition of human and rat GSTs, using 1-chloro-2,4-dinitrobenzene as substrate, was expressed as I25. All compounds caused moderate reversible inhibition (I25 ranged from 0.2 to 5.4 mM for human GSTs and from 0.4 to 4.9 mM for rat GSTs). In rat, eugenol methyl ether was the strongest inhibitor. In human, the overall inhibiting capacities of eugenol, eugenol methyl ether, isoeugenol methyl ether and 4-propyl phenol were more or less similar; 2-allylphenol was the poorest inhibitor.

Effect of eugenol on the genotoxicity of established mutagens in the liver.

The influence of in vivo treatment with eugenol on established mutagens was studied to determine whether eugenol has antigenotoxic potential. The effects of eugenol in rats was investigated in the unscheduled DNA synthesis (UDS) assay with established mutagens and the Salmonella typhimurium mutagenicity assay. In addition, the effect of in vivo treatment with eugenol on benzo[a]pyrene (B[a]P)-induced genotoxicity in human hepatoma cell line Hep G2 was investigated in the single-cell gel electrophoresis assay. The mutagenicity of B[a]P in the S. typhimurium mutagenicity assay was lower in liver S-9 fractions from control rats. Incubation of liver S-9 fractions from eugenol-treated rats with dimethylbenzanthracene (DMBA) had no antimutagenic effect. Eugenol did not modify UDS activity in hepatocytes isolated from rats pretreated with eugenol orally after exposure of these cells in vitro to DMBA and aflatoxin B1. Four different treatment schemes of combinations of B[a]P and eugenol were examined in Hep G2 cells: pre-treatment with eugenol; simultaneous treatment with eugenol and B[a]P; a combination of these (pretreatment/simultaneous treatment); and post-treatment with eugenol. An increase in the genotoxicity of B[a]P was found in Hep G2 cells. No effect of eugenol on the genotoxicity of B[a]P was found with the pre- and post-treatments. It is concluded that the effect of eugenol on genotoxicity induced by established mutagens is not univocal; in vivo treatment of rats with eugenol resulted in a reduction of the mutagenicity of B[a]P in the S. typhimurium mutagenicity assay, while in the UDS assay no effect of eugenol was found. In vitro treatment of cultured cells with eugenol resulted in an increase in genotoxicity of B[a]P. These findings indicate that there is only limited support for the antigenotoxic potential of eugenol in vivo.

Antimutagenicity of eugenol in the rodent bone marrow micronucleus test.

The antimutagenic effect of eugenol on the mutagenicity of cyclophosphamide (CP), mitomycin C (MMC), ethyl methanesulfonate (EMS) and benzo[a]pyrene (B[a]P) was assessed in the rodent bone marrow micronucleus test using male Swiss mice. Oral administration of eugenol (0.4% in the diet) for 15 days was found to decrease significantly the frequency of micronucleated polychromatic erythrocytes (MPEs) elevated by CP. No effect was found on the frequency of MPEs elevated by MMC, EMS and B[a]P. The results provide some support for antimutagenic potency of eugenol in vivo.

Effects of the naturally occurring alkenylbenzenes eugenol and trans-anethole on drug-metabolizing enzymes in the rat liver.

In order to study the effects of trans-anethole and eugenol on drug-metabolizing enzyme activities in vivo, male Wistar rats were treated by gavage with trans-anethole (125 or 250 mg/kg body weight) or eugenol (250, 500 or 1000 mg/kg body weight) daily for 10 days. In liver microsomes and cytosol various phase-I and phase-II biotransformation enzyme activities were determined. No effect on total cytochrome P-450 content in liver microsomes from rats treated with eugenol or trans-anethole was observed. Administration of 1000 mg eugenol/kg body weight, but not the lower doses, significantly increased cytochrome P-450-dependent 7-ethoxy-resorufin O-deethylation (EROD) and 7-pentoxyresorufin O-depentylation (PROD); administration of trans-anethole (125 or 250 mg/kg body weight) did not alter EROD and PROD activities. In rat liver cytosol, UDP-glucuronyl transferase (GT) activity towards the substrate 4-chlorophenol was significantly increased in all treated rats, and activity towards 4-hydroxybiphenyl as substrate was significantly increased in rats treated with 250 mg trans-anethole/kg or with 500 or 1000 mg eugenol/kg. DT-diaphorase (DTD) activity was only significantly enhanced in the liver cytosol of rats treated with trans-anethole at 250 mg/kg body weight. Enhancement of cytosolic glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene was found for all eugenol- and trans-anethole-treated rats. In addition, significantly increased levels of GST subunit 2 were measured by HPLC in the liver cytosol of rats treated with eugenol (500 or 1000 mg/kg body eight) or trans-anethole (250 mg/kg body weight). It is concluded that both eugenol and trans-anethole preferentially induced phase II biotransformation enzymes in rat liver in vivo.