Safety evaluation of pectin-derived acidic oligosaccharides (pAOS): genotoxicity and sub-chronic studies.

Pectin-derived acidic oligosaccharides (pAOS) are non-digestible carbohydrates to be used in infant formulae and medical nutrition. To support its safety, the genotoxic potential of pAOS was evaluated. pAOS was not mutagenic in the Ames test. Positive results were obtained in the chromosome aberration test only at highly cytotoxic concentrations. The effects obtained in the mouse lymphoma test were equivocal; pAOS was not mutagenic in vivo. A sub-chronic dietary study, preceded by 4-week parental and in utero exposure phase, investigated general safety. Administration of pAOS did not affect parental health nor pup characteristics. No effects specific for acidic oligosaccharides were observed in the subsequent sub-chronic study. Slight diffuse hyperplasia of epithelial layer of the urinary bladder was noted to result from concurrently elevated urinary sodium, due to high sodium in pAOS, and elevated urinary pH. This phenomenon was confirmed in a mechanistic (sub-chronic) study. In contrast, in rats fed pAOS in combination with NH(4)Cl, an acidifying agent, the induced low urinary pH completely prevented the development of urothelial hyperplasia. Hyperplasia induced by this mechanism in rats is considered not relevant to man. Based on the current knowledge we consider pAOS safe for human consumption under its intended use.

Androgenic activity in surface water samples detected using the AR-LUX assay: indications for mixture effects.

This paper describes the screening of 22 extracts from 18 different aquatic environmental samples for androgenic activity, including indirect and interactive effects on androgen receptor (AR)-mediated signal transduction, using the AR-LUX bioassay. Four samples, originating from an industrial wastewater treatment plant (WTP) or the river Meuse, were shown to contain substantial androgenic activity. Moreover, the samples originating from the industrial WTP showed an enhancement of the maximal androgenic response relative to that elicited by the standard androgen methyltrienolone (R1881) in the AR-LUX assay. This indicates the involvement of cellular mechanisms other than receptor-ligand interaction influencing AR-regulated pathways. This also demonstrates the additional value of cell based assays featuring a more complete array of fully functional interacting pathways. Chemical analysis using GC-MS confirmed the presence of a number of androgens and also estrogens in these WTP samples. Subsequently, we showed that estrone and tributyltin hydride (TBT-H) enhance the response to androgens. This indicates that the presence of numerous compounds in addition to androgens in environmental mixtures might very well result in a more profound perturbation of the normal physiology of exposed organisms than estimated based on the androgen levels alone. Therefore, risk assessment of environmental samples should include an evaluation of the presence and the interactive effects of (ant)agonists of carefully selected relevant cellular receptors in order to provide a realistic estimate of the integrated ecotoxicological risk of the compounds present.

Developmental exposure to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107): long-term effects on brain development, behavior, and brain stem auditory evoked potentials in rats.

In the present study the developmental neurotoxic effects of the PCB metabolite 4-OH-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107) were compared with effects caused by a mixture of parent polychlorinated biphenyl (PCB) congeners (Aroclor 1254). Pregnant female Wistar rats were exposed to 0.5 or 5 mg 4-OH-CB107, or 25 mg Aroclor 1254 per kg body weight from gestation days 10 to 16. Plasma thyroid hormone levels were significantly decreased in the offspring of all treatment groups at postnatal day 4 (PND 4). Behavioral experiments using an open field paradigm revealed an impaired habituation in male offspring of all treatment groups at PND 130. Passive avoidance experiments indicated significant influences on the time course of step-down latencies across trials in exposed male rats. Catalepsy induced by haloperidol showed increases in latencies to movement onset in female offspring exposed to 0.5 mg 4-OH-CB107 compared to Aroclor 1254 treated offspring at PND 168-175. Male offspring exposed to 4-OH-CB107 or Aroclor 1254 showed decreases in latencies compared to control animals. Brain stem auditory evoked potentials (BAEPs) measured at PND 300-310 showed significant increases in auditory thresholds in the low frequency range between Aroclor 1254 and 4-OH-CB107 (5 mg/kg bw) treated animals. Measurements of neurotransmitter levels revealed effects of Aroclor 154 exposure on both the dopaminergic and the serotonergic systems, whereas 4-OH-CB107 exposure affected dopaminergic and noradrenergic systems, with slight but not significant effects on the serotonergic system. These results indicate that 4-OH-CB107 is able to induce long-term effects on behavior and neurodevelopment. The observed effects for 4-OH-CB107 are similar to, but in some aspects different from, the effects observed after Aroclor 1254 exposure.

Effects of in utero exposure to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107) on developmental landmarks, steroid hormone levels, and female estrous cyclicity in rats.

Previous studies have revealed that one of the major metabolites of PCBs detected in human blood, 4-OH-2,3,3',4',5-pentaCB (4-OH-CB107), accumulated in fetal liver, brain, and plasma and reduced maternal and fetal thyroid hormone levels after prenatal exposure to pregnant rats from gestational days (GD) 10-16. In the present study, the effects of 4-OH-CB-107 on developmental landmarks, steroid hormone levels, and estrous cyclicity of rat offspring after in utero exposure to 4-OH-CB107 was investigated. Pregnant rats were exposed to 0, 0.5, and 5.0 mg 4-OH-CB107 per kg bw from GD 10 to GD 16. Another group of rats was exposed to Aroclor 1254 (25 mg/kg bw) to study the differences between effects caused by parent PCB congeners and the 4-OH-CB107 alone. A significant, dose-dependent prolongation of the estrous cycle was observed in 75% and 82% of female offspring exposed to 0.5 and 5.0 mg 4-OH-PCB107, respectively, compared to 64% of Aroclor 1254 (25 mg/kg) exposed offspring. The diestrous stage of the estrous cycle was prolonged, resembling a state of pseudopregnancy, which might reflect early signs of reproductive senescence. Plasma estradiol concentrations in female rat offspring were significantly increased (50%) in the proestrous stage after exposure to 5 mg 4-OH-CB107 per kg bw. No effects on estradiol levels were observed in Aroclor 1254 treated animals. These results indicate that in utero exposure to 4-OH-CB107 leads to endocrine-disrupting effects, especially in female offspring. The possible impact on neurobehavior following exposure to 4-OH-CB107 will be reported elsewhere.

Placental transfer of a hydroxylated polychlorinated biphenyl and effects on fetal and maternal thyroid hormone homeostasis in the rat.

Earlier studies at our laboratory indicated that several hydroxylated polychlorinated biphenyls (OH-PCBs) detected in human blood could specifically inhibit thyroxine (T(4)) transport by competitive binding to the thyroid hormone transport protein transthyretin (TTR) in vitro. In the present study we investigated the effects of prenatal exposure to 5 mg/kg body weight of [14C]-labeled or unlabeled 4-OH-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107), one of the major metabolites of PCBs detected in human blood, from gestation days (GD) 10 to 16 on thyroid hormone status and metabolism in pregnant rats and their fetuses at GD 17 and GD 20. 4-OH-CB107 is a metabolite of both 2,3,3',4,4'-pentachlorobiphenyl (CB-105) and 2,3',4,4',5-pentachlorobiphenyl (CB-118). We were able to show the accumulation of 4-OH-CB107 in the fetal compartment. The fetal/maternal ratios at GD 20 in liver, cerebellum, and plasma were 11.0, 2.6, and 1.2, respectively. The 14C-4-OH-CB107-derived radioactivity in plasma was bound to TTR in both dams and fetuses. Fetal plasma TT(4) and FT(4) levels were significantly decreased at GD 17 and GD 20 (89% and 41% respectively at GD 20). Fetal thyroid stimulating hormone levels were increased by 124% at GD 20. The T(4) concentrations in fetal forebrain homogenates at GD20 were reduced by 35%, but no effects could be detected on brain T(3) concentrations. The deiodination of T(4) to T(3) was significantly increased in fetal forebrain homogenates at GD 17, and unaltered at GD 20. In addition, no alterations were observed in maternal and fetal hepatic T(4)-UDP-glucuronosyltransferase activity, type I deiodinase activity, and EROD activity. In conclusion, exposure of pregnant rats to 4-OH-CB107 results in the distribution of the compound in the maternal and fetal compartment, which is probably caused by the binding of the PCB metabolite to TTR. Consequently, TT(4) levels in fetal plasma and brain samples were reduced. Despite reductions in fetal brain T(4) levels, the active hormone (T(3)) in fetal brains remained unaffected.

Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location.

In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between and were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in than in and . Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in . Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to any particular pesticides. Besides, the contribution of non-analyzed xeno-estrogens as alkylphenol(-ethoxylates) and bisphenol-A to the estrogenic potency of rainwater could not be excluded. Further research should focus on the chemical identification of estrogenic compounds in rainwater. In addition, more attention should be given to the ecological consequences of atmospheric deposition of individual pesticides and of total toxic potencies that regularly exceed environmental criteria for Dutch surface waters and/or toxic threshold values for aquatic organisms.

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

Embryotoxic potential of persistent organic pollutants extracted from tissues of guillemots (Uria aalge) from the Baltic Sea and the Atlantic Ocean.

The Baltic Sea is a heavily polluted area. To assess the current contaminant pressure on the common guillemot (Uria aalge) living there, whole-body extracts of guillemots from the Baltic Sea were prepared and subdivided over six fractions, which differed in composition due to lipophilicity and polarity of the contaminants. The fractions were tested in the chicken embryo assay and compared to fractions of Atlantic guillemot extracts. Fertilized chicken eggs were injected with 0.03, 0.3, or 3 bird egg equivalents (BEQ) of the contaminants present in the fractions and then incubated for 19 d. Endpoints were selected to cover several mechanisms that may play a role in reproductive failures of fish-eating birds. Fractions I and IV from the Baltic guillemots induced ethoxyresorufin-O-deethylase (EROD) activity up to 15-fold in embryos exposed to 0.3 BEQ and up to 17-fold in embryos exposed to 3 BEQ. Corresponding Atlantic fractions induced EROD activity only at the higher dose of 3 BEQ. Morphological alterations were observed in the bursa of Fabricius in embryos exposed to the fractions that induced EROD, and for the Baltic fractions, this was apparent at the dose of 0.3 BEQ. The higher toxic potency of fractions I and IV was confirmed by higher mortality and occurrence of malformations among embryos exposed to these fractions. No other effects were observed; morphometry, hepatic porphyrin levels, thiamine-dependent enzymes, and acetylcholinesterase activity were not affected by any fraction. During interpretation of the results, concentrations in the whole-body guillemot extracts were compared to concentrations reported in field studies. In general, concentrations in the guillemot extract were lower than those associated with biomarker responses in other wild-bird species. However, because the relative sensitivity of guillemot toward immunotoxic effects remains to be resolved, effects on the immunocompetence of guillemot could not be excluded.

Imposex induction in laboratory reared juvenile Buccinum undatum by tributyltin (TBT).

Here we report a series of experiments on the development and occurrence of imposex in the common whelk, Buccinum undatum, under the influence of (chronic) exposure to butyltin compounds. The main objective of the experiments was to obtain more information about the effects of organotin compounds in the marine environment, which possibly relate to the reported decline of B. undatum in Dutch coastal waters. In these studies tributyltin (TBT) dose-dependently induced the development of male sexual organs in juvenile whelks. A TBT concentration >7 ng Sn/l induced imposex in juvenile whelks. Growth in TBT-exposed juvenile whelks was significantly reduced compared to the reference group at a nominal TBT dose ≥ 4 ng Sn/l in one of the exposure studies. After 5 years in the laboratory, egg-laying was only observed in reference aquaria. Thus, TBT might impair whelk reproduction through growth reduction. The results showed a sensitivity towards imposex development in different life-stages. Juveniles were the most sensitive, adolescent females also responded, but adult females did not respond to TBT exposure, although they dose-dependently increased their organotin (OT) body-burden when exposed. Environmental TBT during only the in ovo stage, did not result in an increased masculinisation compared to non-exposed developing whelks. Histological studies showed no sterilisation due to mechanical blockage of the (adult) female genital opening by sperm-duct tissue. Gonadal development in 2-year old juveniles was not observed. This implies that the differentiation of a penis and a vas deferens, which already occurred in the first few months after hatching, was not controlled by gonadal factors. No other sexual characteristics than those already visible with the eye were found. TBT inactivated CYP450 to its inactive form CYP420 in in vitro exposure studies with microsomal fractions of whelks. The studies have shown TBT to disrupt sexual development dose dependently in juvenile common whelks. TBT also dose dependently exerts an effect on enzymatic (CYP450) processes. Although no mechanical sterilisation was observed, reproduction might be impaired through growth reduction.

The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids.

Natural antioxidants like vitamin C, vitamin E, carotenoids, and polyphenols like flavonoids, are at present generally considered to be beneficial components from fruit and vegetables. The anti-oxidative properties of these compounds are often claimed to be responsible for various beneficial health effects of these food ingredients. Together these studies provide the basis for the present rapidly increasing interest for the use of natural antioxidants as functional food ingredients and/or as food supplements. However, at higher doses or under certain conditions antioxidant-type functional food ingredients may exert toxic pro-oxidant activities. The present manuscript gives an overview of especially this pro-oxidative chemistry and toxicity of well-known natural antioxidants including vitamin C, vitamin E, carotenoids and flavonoids.

Botanical health products, positioning and requirements for effective and safe use.

Within the group of botanical products there is a large range of variation with regard to their properties. Some products are identical to foods while others come close to or are medicines. Botanical products are regulated differently within the different member states of the European Union (EU) and globally. They are regulated either as food or as medicinal products, and in the latter case often with simplified registration procedures. These differences are caused by differences in traditional use, in cultural and historical background, in scientific substantiation and in enforcement of current legislation. One may expect that in the future differences will remain, unless EU legislation is enacted with sufficient room for different approaches. The strengths and weaknesses of the different regulatory procedures have been reviewed and evaluated as well as the current methods for quality, efficacy and safety evaluation. Criteria to categorize botanical products have been defined, such that botanical products can be regulated under the current food and medicinal regulations. Furthermore, a decision tree has been developed as a tool to distinguish herbal medicinal products from botanical health products and vice versa, and to provide a stepwise framework for the assessment of safety and efficacy.